Corticosteroid -Encapsulated Nanoparticles in ...
Post on 25-Oct-2021
9 Views
Preview:
Transcript
Corticosteroid-Encapsulated Nanoparticles in Thermoreversible Gels for the Amelioration of Choroidal Neovascularization in Age-Related Macular Degeneration
Anjali Hirani
Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirement for the degree of
Doctor of Philosophy in
Biomedical Engineering
Yong W. Lee, Chair Luke E. Achenie
Aaron S. Goldstein Liwu Li
Vijaykumar B. Sutariya Yashwant Pathak
April 1, 2015 Blacksburg, VA
Keywords: Choroidal Neovascularization, Age-Related Macular Degeneration, Sustained Drug Delivery
Corticosteroid-Encapsulated Nanoparticles in Thermoreversible Gels for the Amelioration of Choroidal Neovascularization in Age-
Related Macular Degeneration
Anjali Hirani
Abstract Age-related macular degeneration (AMD) is one of the leading causes of blindness in
adults over the age of 60. Currently, at least 11 million patients in the United States have some
form of macular degeneration and this number is projected to grow as the population ages. The
more severe form of the disease – neovascular (wet) AMD, is characterized by intraocular
neovascularization, inflammation, and retinal damage; however, the disease progression can be
deterred through intraocular injections of anti-angiogenic agents. The complications and burden
that arise from repetitive injections as well as the difficulty posed by targeting the posterior
segment of the eye make this an interesting territory for the development of novel drug delivery
systems. New methods for drug delivery are being investigated exploring the use of
nanoparticles and other polymeric materials.
The goal of this project is to study the potential use of poly(lactide-co-glycolic acid)-
polyethylene glycol (PLGA-PEG) nanoparticles in thermoreversible gels as localized sustained
intraocular drug delivery. We prepared stable and reproducible corticosteroid-encapsulated
nanoparticles in thermoreversible gels to inhibit vascular endothelial growth factor (VEGF)
overexpression characteristic of neovascular AMD. We characterized the drug delivery system
by obtaining size, shape, and drug encapsulation data. We also demonstrated that the polymer
could be injected into the vitreous as a solution and transition to a gel phase based on the
temperature difference between regular indoor environment and the vitreous body. The drug
delivery system was tested on human retinal pigment epithelial cells (ARPE-19), for
cytotoxicity, uptake and VEGF expression.
We also examined the drug delivery system’s ability to mitigate the disease progression
in a mouse model of choroidal neovascularization (CNV). The effect on blood vessel area was
shown and the changes in the mRNA expression of angiogenesis mediators were analyzed by
iii
real-time reverse transcription polymerase chain reaction (RT-PCR). These results indicate that
the proposed drug delivery systems has the promise to be developed for retinal diseases,
involving CNV, including neovascular AMD. Further studies are warranted in developing this
promising intraocular drug delivery system for wet AMD and similar ophthalmic diseases.
iv
Acknowledgements
This journey has been carried out with incalculable support and advice from numerous
individuals. I would first like to express my gratitude and respect to my advisor, Dr. Yong Woo
Lee, for your guidance and patience throughout my graduate studies. I will always admire your
scientific expertise and analytical skills. Everything I have achieved is due to your influence and
support.
I would like to acknowledge my committee members for their insight and their individual
efforts in helping me through this process. I could not have completed this work without the help
and expertise of Dr. Yashwant Pathak. You have taught me the true meaning of a guru and gave
me a way to follow my dreams when I thought there were no other options available. I will
always be grateful to you. Dr. Sutariya, thank you for opening your lab to me and giving me a
position here. You have given me so many opportunities in such a short time and so much of the
progress I made is due to you. You have reignited my interest in research and I have looked
forward to coming in and working each and every day.
Dr. Luke Achenie, I have learned so much from you on other projects over the past several
years. Your passion for science and academia is inspiring. Dr. Aaron Goldstein, thank you for
your willingness to guide me in the projects I have worked on over the years. It has been a
pleasure to learn from you. Dr. Liwu Li, you are truly an expert in your field and I appreciate
your advice.
Additionally, another professor who provided tremendous support is Dr. Radouil Tzekov.
Your willingness to take time out of your hectic schedule to teach a student who blindly reached
out to you is humbling. It has truly been a pleasure working with you and I will aspire to guide
and connect with others as you have done with me.
I would like to thank my fellow lab members, both at Virginia Tech and USF, who have
become family to me: Hyung Joon Cho, Won Hee Lee, Aditya Grover, Aum Solanki, Chia Tha
Thach and Xingxiao (Shelly) Li. We have created a dynamic environment together and it has
been a joy to work with you on a daily basis.
No degree could be completed successfully without the support of the administrative staff
and I am especially grateful to Tess Sentelle and Pam Stiff who have both been so helpful and
cheerful along the way.
v
Most importantly, I need to thank my sister, parents and extended family members. You have
supported me from the beginning, prayed for me, pushed me and even pulled me back on track
when I was discouraged. My mother and father have been my rock and inspiration. I am so
blessed to have my sister, Arti, in my life. Everyone should be so lucky to have their own
personal cheerleader dancing alongside them in their journey.
vi
Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iv Table of Contents ........................................................................................................................... vi List of Abbreviations ..................................................................................................................... ix List of Figures and Tables............................................................................................................... x Chapter 1. Introduction ................................................................................................................... 1
1.1 Problem Statement .......................................................................................................... 1 1.2. Background and Significance .............................................................................................. 2
1.2.1 Age-related macular degeneration ................................................................................. 2 1.2.2 Vascular endothelial growth factor (VEGF) .................................................................. 2 1.2.3 Current therapies and challenges ................................................................................... 3 1.2.4 Routes of drug administration ........................................................................................ 6 1.2.5 Drug delivery systems for posterior segment ocular disorders ...................................... 6
1.3 Hypothesis and Specific Aims ............................................................................................ 10 Chapter 2. Triamcinolone Acetonide Nanoparticles Incorporated in Thermoreversible Gels for Age-Related Macular Degeneration ............................................................................................. 11
2.1. Abstract .............................................................................................................................. 11 2.2. Introduction ........................................................................................................................ 12 2.3. Materials and Methods ....................................................................................................... 15
2.3.1. Materials ..................................................................................................................... 15 2.3.2. Nanoparticle synthesis ................................................................................................ 15 2.3.3. Nanoparticle characterization ..................................................................................... 16 2.3.4. Drug encapsulation efficiency .................................................................................... 16 2.3.5. Preparation of thermoreversible gels .......................................................................... 16 2.3.6. Determination of gelation temperature ....................................................................... 16 2.3.7. In vitro release............................................................................................................. 17 2.3.8. Cell culture .................................................................................................................. 17 2.3.9. Cytotoxicity................................................................................................................. 17 2.3.10. VEGF secretion ......................................................................................................... 18 2.3.11. Statistical analysis ..................................................................................................... 18
2.4. Results ................................................................................................................................ 18 2.4.1. Nanoparticle characterization ..................................................................................... 18 2.4.2. Drug encapsulation efficiency .................................................................................... 19 2.4.3. Determination of gelation temperature ....................................................................... 19 2.4.4. In vitro release............................................................................................................. 21 2.4.5. Cytotoxicity................................................................................................................. 22 2.4.6. VEGF secretion ........................................................................................................... 23
2.5. Discussion .......................................................................................................................... 25 2.6. Conclusion ......................................................................................................................... 27
Chapter 3. Efficacy of Loteprednol Etabonate Drug Delivery System in Suppression of in vitro Retinal Pigment Epithelium Activation ........................................................................................ 28
3.1 Abstract ............................................................................................................................... 28 3.2 Introduction ......................................................................................................................... 29 3.3 Materials and Methods ........................................................................................................ 31
vii
3.3.1 Materials ...................................................................................................................... 31 3.3.2 Nanoparticle synthesis ................................................................................................. 32 3.3.3 Nanoparticle characterization ...................................................................................... 32 3.3.4 Drug entrapment efficiency ......................................................................................... 32 3.3.5 Scanning electron microscopy (SEM) ......................................................................... 33 3.3.6 Preparation of thermoreversible gels ........................................................................... 33 3.3.7 In vitro release.............................................................................................................. 33 3.3.8 Cell culture ................................................................................................................... 34 3.3.9 Cytotoxicity.................................................................................................................. 34 3.3.10 Intracellular uptake of coumarin-6-loaded NPs in ARPE-19 cells ............................ 35 3.3.11 VEGF secretion .......................................................................................................... 35 3.3.12 Statistical analysis ...................................................................................................... 36
3.4 Results ................................................................................................................................. 36 3.4.1 Nanoparticle characterization ...................................................................................... 36 3.4.2 Drug entrapment efficiency ......................................................................................... 36 3.4.3 Scanning electron microscopy (SEM) analysis of NPs formulations .......................... 37 3.4.4 In vitro release............................................................................................................. 37 3.4.5 Cytotoxicity.................................................................................................................. 38 3.4.6 Intracellular uptake of coumarin-6-loaded NPs ........................................................... 39 3.4.7 VEGF secretion study .................................................................................................. 40
3.5 Discussion ........................................................................................................................... 42 3.6 Conclusions ......................................................................................................................... 44
Chapter 4. The Effect of Corticosteroid-Nanoparticles Incorporated in a Thermoreversible Gel on Chemically Induced Choroidal Neovascularization in Mice ........................................................ 45
4.1 Introduction ......................................................................................................................... 45 4.2 Materials and Methods ........................................................................................................ 46
4.2.1 Animals ........................................................................................................................ 46 4.2.2 Study design ................................................................................................................. 46 4.2.3 PEG-induced choroidal neovascularization ................................................................. 46 4.2.4 Treatment administration ............................................................................................. 46 4.2.5 Preparation of flat mounts and CNV evaluation .......................................................... 47 4.2.6 Real-time reverse transcription-polymerase chain reaction (RT-PCR) ....................... 47 4.2.7 Statistical analysis ........................................................................................................ 47
4.3 Results ................................................................................................................................. 48 4.3.1 CNV induction ............................................................................................................. 48 4.3.2 Effect of corticosteroid drug delivery systems on CNV area ...................................... 49 4.3.3 Effect of corticosteroid drug delivery systems on VEGF expression .......................... 53 4.4 Discussion ....................................................................................................................... 53 4.5 Conclusions ..................................................................................................................... 55
Chapter 5. Conclusions and Future Work ..................................................................................... 57 5.1. Summary and Conclusions ........................................................................................... 57 5.2. Future Directions .......................................................................................................... 58
5.2.1. Effect of factors that would alter drug release from DDS .................................... 58 5.2.2. Effect of DDS on molecular mechanisms of CNV ............................................... 59 5.2.3. Combination therapy with corticosteroid DDS and anti-vascular endothelial growth factor (VEGF) for an enhanced anti-angiogenic effect ............................................ 59
viii
References ..................................................................................................................................... 60 Appendix A: Ocular Toxicity of Nanoparticles ............................................................................ 69 Appendix B: Nanotechnology for Omics-based Ocular Drug Delivery ....................................... 81
ix
List of Abbreviations
AMD: Age-related macular degeneration
BRB: Blood retinal barrier
CNV: Choroidal neovascularization
DDS: Drug delivery system
EE: Encapsulation efficiency
ELISA: Enzyme linked immunosorbent assay
ICAM-1: Intercellular adhesion molecule-1
LE: Loteprednol etabonate
NP: Nanoparticle
PBS: phosphate-buffered saline
PDI: Polydispersity index
PDT: Photodynamic laser therapy
PEG: Polyethylene glycol
PLGA: Poly(lactide-co-glycolic acid)
RPE: Retinal pigment epithelium
RT-PCR: Reverse transcription polymerase chain reaction
TA: Triamcinolone acetonide
TEM: Transmission electron microscopy
VEGF: Vascular endothelial growth factor
x
List of Figures and Tables Figure 1.1: Cross section of macula at normal state (left) and with wet AMD (right) (Medical illustration courtesy of Macular Degeneration Research, a BrightFocus Foundation program (8)). ........................................................... 2 Table 1.1: Corticosteroid comparison chart (32) .......................................................................................................... 4 Table 2.1: NP characterization data carried out in triplicates (n=3) and represented as the mean value ± SD. ....... 18 Figure 2.1: Temperature-dependent phase transitions in PLGA-PEG-PLGA thermoreversible gels based on w/v concentration of gel in aqueous solution. The 20% w/v thermoreversible gel was chosen for further studies because its phase transition occurs over physiologically relevant temperatures. ..................................................................... 20 Figure 2.2: Pictorial representation of the temperature-dependent phase change exhibited by the 20% w/v PLGA-PEG-PLGA thermoreversible gel. The gel solution exists in the liquid state at 4o C and transitions into a gel state at 37o C. ........................................................................................................................................................................... 21 Figure 2.3: In vitro release data of the TA NP and TA NP Gel as compared to equal concentrations of the TA drug. Samples were analyzed by UV spectroscopy (240 nm, λmax) at predetermined intervals over a 10-day period (n=3, mean value ± SD). ....................................................................................................................................................... 22 Figure 2.4: MTT cytotoxicity data in ARPE-19 cells of equal treatments (10 μM) of the following: Blank NP, TA free drug, TA NPs, TA drug in 20% w/v TR gel, and TA NPs in 20% w/v TR gel as compared to untreated control cells. Experiments were carried out in statistical triplicates (n=3) and quantified by absorbance reading at 570 nm (Synergy H4 Plate reader, Biotek Industries Inc.). Data is represented as mean number of viable cells ± SD. Statistical tests were carried out using paired t-test (p≤0.05). .................................................................................... 23 Figure 2.5: Time-dependent inhibition of VEGF secretion in ARPE-19 cells through equal concentration treatments (100 μM) of blank NPs, TA free drug, and TA NPs at 12 and 72 hours. Experiments were carried out using the ELISA method (Human VEGFA ELISA kit, Thermo Scientific) in statistical triplicates (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD. ................................................................... 24 Figure 2.6: Comparative suppression of VEGF secretion in ARPE-19 cells through equal concentration treatments (10 μM) of TA free drug, TA NPs, TA drug in 20% w/v TR gel, and TA NPs in 20% w/v TR gel at 72 hours. Experiments were carried out using the ELISA method (Human VEGFA ELISA kit, Thermo Scientific) in statistical triplicates (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD. . 25 Table 3.1: Nanoparticle (NP) characterization data of particle size and polydispersity index (PDI) carried out in triplicates (n=3) and represented as the mean value ± SD. ........................................................................................ 36 Figure 3.1: SEM visualization of loteprednol etabonate-loaded nanoparticles shows round morphology. Samples were diluted 1:10 and visualized by SEM. Samples were read at 15,000x magnification and 5kV acceleration voltage. ........................................................................................................................................................................ 37 Figure 3.2: In vitro release data of the loteprednol etabonate nanoparticles (LE NP) compared to LE NP gel. Samples were analyzed by UV spectroscopy (243 nm, λmax) at predetermined intervals over a 7-day period (n=3, mean value ± SD). ....................................................................................................................................................... 38 Figure 3.3: MTT cytotoxicity data in ARPE-19 cells of increasing concentrations of loteprednol etabonate (LE) free drug, LE NPs, and LE NPs in 20% w/v TR gel as compared to untreated control cells. Experiments were carried out in triplicates (n=3) and quantified by absorbance reading at 570 nm. Data is represented as mean ± SD. ............... 39 Figure 3.4: Cellular uptake of coumarin-6-loaded NPs in ARPE-19 cells. Nuclei were stained with DAPI visible in first panel. The uptake of coumarin-6-loaded NPs is depicted in second panel. Membrane staining with Cell Mask Deep Red is shown in the third panel. The final panel displays the overlaying images. Magnification of 60x. .......... 40 Figure 3.5: Comparative suppression of VEGF secretion in ARPE-19 cells through increasing concentrations (1, 10, μM) of loteprednol etabonate (LE) free drug, LE NPs, and LE NP gel at 12 hours. Experiments were carried out using the ELISA method (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD. *p<0.05 vs. Control. ................................................................................................................................ 41 Figure 3.6: Comparative suppression of VEGF secretion in ARPE-19 cells through equal concentration treatments (10 μM) of LE free drug, LE NPs, and LE NPs in 20% w/v TR gel at 72 hours. Experiments were carried out using the ELISA method (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD. *p<0.05 vs. Control. ............................................................................................................................................. 42 Figure 4.1: (A) FITC-dextran labeled retinal/choroidal flat-mount. Control (left) and CNV-induced eye (right). Magnification of 20x. (B) Total area of blood vessels in control and CNV-induced eye. Data is represented by mean ± SD (n=3). *p<0.05. .................................................................................................................................................. 48 Figure 4.2: Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area at 2 weeks. Groups tested include CNV-induced (no treatment), loteprednol etabonate (LE),
xi
LE drug delivery system (DDS), triamcinolone acetonide (TA), TA DDS. Data is represented by mean ± SD (n=3). *p<0.05 vs CNV. ......................................................................................................................................................... 49 Figure 4.3: Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area at 4 weeks. Groups tested include CNV-induced (no treatment), loteprednol etabonate (LE), LE drug delivery system (DDS), triamcinolone acetonide (TA), TA DDS. Data is represented by mean ± SD (n=3). *p<0.05 vs. CNV. ........................................................................................................................................................ 50 Figure 4.4: Comparison of sustained efficacy of LE and LE DDS on CNV area at 2 and 4 weeks. Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area. Data is represented by mean ± SD (n=3). *p<0.05. ................................................................................................................ 51 Figure 4.5: Comparison of sustained efficacy of TA and TA DDS on CNV area at 2 and 4 weeks. Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area. Data is represented by mean ± SD (n=3). *p<0.05. ................................................................................................................ 52 Figure 4.6: Effect of corticosteroids and corticosteroid drug delivery systems on VEGF expression at 4 weeks. Data is represented by mean ± SD (n=3). *p<0.05 vs. CNV. .............................................................................................. 53
1
Chapter 1. Introduction
1.1 Problem Statement
Based on the 2010 U.S. Census, it was estimated that 38.2 million people over the age of 40
are affected by vision loss due to ocular diseases in the United States (1). Age-related macular
degeneration (AMD) affects the posterior segment of the eye. Specifically, the central part of the
retina, responsible for the subject’s ability to see fine details – the macule, is damaged by
abnormal growth of blood vessels in the choroid which can penetrate into the retina itself. These
newly-formed blood vessels have increased permeability and that leads to sub- and/or intestinal
edema which could affect negatively central vision (2). While the exact cause is unknown, lack
of nutrient supply for the macula and UV exposure are believed to contribute to the progression.
Therapeutic intervention is limited due to the physiological barriers of the eye that limit potential
routes of drug administration (3).
While drugs are available clinically to delay or even stop the progression of posterior
segment ocular diseases, they are limited by the need for repeated intravitreal administration to
maintain the therapeutic levels. The development of new drugs is time consuming and expensive;
therefore, more efficient and safer drug delivery systems would benefit disease treatment.
Thermoreversible hydrogels are a unique delivery vehicle for the eye. They are a type of in situ
gels that can be administered as a solution and undergo gelation with a change in temperature (4,
5). PLGA-PEG-PLGA thermoreversible polymers can be tuned to induce gelation at body
temperature (6). Additionally, nanoparticles incorporated in thermoreversible gel for use in drug
delivery offer novel strategies for sustained intraocular delivery.
2
1.2. Background and Significance
1.2.1 Age-related macular degeneration
Age-related macular degeneration (AMD) is a sight-threatening disease that affects
central vision. It is localized mostly in the central region of the retina known as the macula,
which is responsible for fine vision. One of the main characteristics of AMD is the formation of
subretinal deposits, called drusen. Advanced AMD can be evolve into two forms – a non-
neovascular (‘dry’) form, characterized by an atrophy of the RPE and outer retina (‘geographic
atrophy’) and a neovascular (‘wet’) form, characterized by choroidal neovascularization (CNV),
the growth of abnormal blood vessels below or extending into the retina (7). Defects in the
membrane separating the (retinal pigment epithelium) RPE and choroid (Bruch’s membrane)
could lead to fluid and blood leakage into the subretinal space, leading to irregularities of the
retina structure and affecting retinal function (Figure 1.1).
Figure 1.1: Cross section of macula at normal state (left) and with wet AMD (right) (Medical illustration courtesy of Macular Degeneration Research, a BrightFocus Foundation program (8)).
1.2.2 Vascular endothelial growth factor (VEGF)
Due to the neovascularization present in AMD, anti-angiogenic therapy is useful to slow
the progression of the disease (9, 10). Vascular endothelial growth factor (VEGF) is recognized
for its role in the pathogenesis of neovascular AMD as a promoter of angiogenesis and vascular
permeability (11-13). The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D,
VEGF-E, as well as placenta growth factor (PIGF) (14). VEGF-A is the prototype member and
3
commonly referred to as VEGF. VEGF is produced by many sources, including endothelial cells,
photoreceptors and RPE (14). VEGF levels in the vitreous are elevated in human CNV when
compared to healthy controls (14). Elevated VEGF levels lead to the breakdown of the blood
retinal barrier (BRB) (13, 15) and can also increase inflammation via induction of inflammatory
mediators like intercellular adhesion molecule-1 (ICAM-1). Therefore, VEGF is a potential
pharmaceutical target for the treatment of AMD (16-18).
1.2.3 Current therapies and challenges
1.2.3.1 Anti-angiogenic drugs
Due to the implication of VEGF in the progression of AMD, anti-angiogenic drugs have
been recently pursued to block the development and leakage of newly formed, abnormal blood
vessels. Three anti-angiogenic drugs are currently used in the treatment of neovascular AMD:
ranibizumab (Lucentis, Genentech Inc., San Francisco, CA), bevacizumab (Avastin, Genentech
Inc.), and pegaptanib sodium injection (Macugen, OSI Pharmaceuticals Inc., Melville, NY) (7).
Ranibizumab is a human recombinant antibody fragment that displays high binding affinity
towards all VEGF-A isoforms. Clinical trials have shown that ranibizumab helps maintain stable
vision without further progression of the disease in many patients with wet AMD; however,
because of the high cost of the drug, the use of the drug worldwide is limited (19). Bevacizumab
is a recombinant humanized monoclonal antibody, which binds all VEGF-A isoforms. It is FDA
approved for colorectal, lung, and breast cancer, but is used in clinical trials for AMD and in
clinical practice off-label due to lower cost. Pegaptanib is a pegylated aptamer, a single strand of
nucleic acid that binds with specificity to a particular target, in this case, VEGF and thus, acts as
an anti-VEGF agent. It binds the VEGF165 isoform and inhibits angiogenesis, although less
effectively than either bevacizumab or ranibizumab, which led to a considerable decline in use in
recent years (20, 21). Several large, randomized, multicenter clinical trial have demonstrated that
the other two drugs, bevacizumab and ranibizumab, although having different molecular
structure and pharmacokinetic profile, show equivalent efficacy and safety profiles in AMD (22)
and in diabetic macular edema (23). For these anti-angiogenic drugs, the biggest challenges are
the route of administration and duration of action. Intravitreal injections allow for the most direct
approach; however, the chronic nature of the disease requires repeated injections for the rest of
4
the patient’s life, resulting in rare, but sight-threatening side-effects such as retinal detachment,
endophthalmitis and cataract formation (24).
1.2.3.2 Corticosteroids
Corticosteroids are commonly used for treatment of various ophthalmic diseases due to
their anti-inflammatory (25, 26), angiostatic, antipermeable and antifibrotic properties (27). Two
of the most common used corticosteroids, especially for treatment of posterior ocular
inflammatory diseases are triamcinolone acetonide and dexamethasone, often used in
combination with other treatments. They act by binding steroid receptors in cells to induce or
repress targeted genes, thereby inhibiting inflammatory symptoms like edema and vascular
permeability (28). Corticosteroids act on VEGF by inhibiting both VEGF secretion, formation of
prostaglandins, and cytokines (IL-1, IL-3, TNF-α) (26, 29). Corticosteroids can also inhibit other
pro-angiogenic and inflammatory factors, like basic fibroblast growth factor, transforming
growth factor-beta, and ICAM-1 and decrease VEGF levels that are evident in
neovascularization (30, 31).
Ophthalmic steroids are rated by the potency of their anti-inflammatory properties. Table
1.1 gives a comparison chart of common steroids used for ophthalmic purposes.
Table 1.1: Corticosteroid comparison chart (32)
Steroid Equivalent
Glucocorticoid Dose (mg)
Anti-inflammatory
potency relative to
Hydrocortisone
Duration of action (hours)
Half-life
Low potency
Cortisone 25 0.8 8-12
Hydrocortisone 20 1 8-12
Upper mid strength potency
Prednisone 5 4 12-36
Prednisolone 5 4 12-36
5
Triamcinolone Acetonide 4 5 12-36
High potency
Dexamethasone .75 25 36-54
Despite their wide clinical use, steroids can have some drawbacks. High doses of steroids can
lead to adverse effects such as raised intraocular pressure and cataract formation, the later due to
formation of covalent adducts with steroid and lysine residues on the lens capsule (33).
1.2.3.3. Laser therapy
Thermal laser therapy uses a target laser beam to destroy CNV formation by
photocoagulation. The laser energy is absorbed mainly by melanin pigment granules in RPE
cells. Thermal energy released by the heating of the melanin granules coagulates and destroys
surrounding CNV areas. Clinical trials have shown the efficacy of this treatment for sub-foveal
CNV with patients showing a reduction in risk of vision loss (15). While this treatment is simple
and relatively inexpensive, bystander RPE cells and overlying photoreceptors also get damaged,
and relatively high rates of recurrence have been observed (15).
1.2.3.4. Photodynamic laser therapy (PDT)
Verteporfin (Visudyne) is a light-sensitive drug that is absorbed by abnormal blood
vessels. The dye is activated with a low-intensity laser light (wavelength 689 nm). In the
illuminated area, the drug generates highly reactive short-lived singlet oxygen and free radicals
that damage abnormal vessels while having a relatively small effect on the overlaying RPE and
retina. However, it was found that when applied in full fluency, the damaging effect is not
limited to the treated area and the newly formed blood vessels only, but can extend beyond it and
affect the healthy nearby retina (34). This led to the instruction of protocols of reduced laser
intensity (reduced-fluence) photodynamic therapy (35). Furthermore, the treatment is expensive
and side effects include transient visual disturbances, adverse effects at site of injection, and
photosensitivity reactions (13). Additionally, eyes with larger CNV lesions and good visual
acuity do not benefit from PDT (13).
6
1.2.4 Routes of drug administration
The posterior segment of the eye consists of the retina, vitreous, choroid and sclera.
While the anterior segment of the eye can be readily accessed for topical treatment, multiple
physical barriers and clearance mechanisms prevent easy access to the posterior segment. The
topical route is a convenient method of drug delivery; however, there is poor bioavailability due
to nasolacrimal drainage and systemic absorption (36). A model of transient diffusion has shown
that less than 5% of a lipophilic drug and 0.5% of a hydrophilic drug reach the anterior chamber
(37). The amount of available drug transported further decreases across the sclera, choroid, and
retinal pigment epithelium (RPE) (38). Permeability via sclera is reduced with cationic and
lipophilic solutes, and the RPE has tight intercellular junctions for hydrophilic molecules (38).
Additionally, the lymphatic system, blood vessels and active transporters all work to clear drugs
administered through transscleral routes. Drug delivery via systemic routes requires high doses
to obtain a therapeutic concentration in the posterior eye due to the tight barrier of the RPE.
Intravitreal injections circumvent physiological barriers and maintain therapeutic doses without
damage to bystander tissues. However, due to the liquefaction of the vitreous body related to
aging, drug delivery can be non-uniform across different retinal areas (39). Furthermore,
frequent injections can lead to sight-threatening complications like retinal detachment, increase
in intraocular pressure, hemorrhage and endophthalmitis (40). Given the presence of these
physiological barriers, the development of therapies that efficiently deliver drugs and extend
drug release to the posterior segment of the eye would be beneficial to the progression of ocular
disease treatment.
1.2.5 Drug delivery systems for posterior segment ocular disorders
Age-related macular degeneration is one of the most prevalent posterior segment
disorders and its chronic nature results in a challenging drug delivery problem. It currently
requires multiple injections to maintain therapeutic concentrations, which can increase the risk of
sight-threatening complications. Sustained drug delivery devices offer alternatives to reduce the
frequency of administration and, as a result the burden on the patient and the physician. The use
of drug delivery systems (DDS) provides an effective method to deliver drugs to the posterior
segment of the eye for extended periods of time (several months or even years). By preparing
7
drug/polymer ophthalmic formulations, drug release can be controlled over an extended duration
(41). For any DDS therapy to be effective, it should fulfill three major goals:
1. Drug release must be targeted to the site of action,
2. Drug release should be controlled at an optimal rate to reduce toxicity,
3. Drug should maintain therapeutic efficacy at adequate dosage levels to eliminate need for
repeated administration (42).
Drug delivery systems can provide strategies to circumvent physiological barriers and
provide sustained release with minimal systemic side effects, thereby expanding current disease
therapy and repurposing presently used drugs and extending their patent life. Several DDS being
investigated to enhance therapeutic profiles of current drug therapies are described below.
1.2.5.1 Ocular implants
Recently, ocular implants have been used to provide platforms for sustained release of
steroids. They are implanted either into the vitreous or on the sclera for intravitreal or transscleral
delivery. Biodegradable implants degrade in the eye and do not require surgical removal;
however, the degradation process can result in inconsistent drug release profiles (3). OzurdexTM
(Allergan Inc. Irvine, CA) is composed of poly(lactide-co-glycolic acid) (PLGA) and releases
dexamethasone intravitreally over a period of 4 to 6 weeks. Currently, this DDS is approved by
FDA for macular edema following branch retinal vein occlusion, central retinal vein occlusion,
for diabetic macular edema and for uveitis. Results have shown an improvement in intraocular
inflammation for up to six months; however, surgery is required for placement and removal of
the implant (3, 43). Non-biodegradable implants provide more accurate control of drug release,
but also require surgical removal. Polymers such as silicone, polyvinyl alcohol, and ethylene
vinyl acetate are typically used in these implants (3). Several implants are in clinical use and
clinical trials; however, studies have shown that while they reduce disease symptoms, they
increase intraocular pressure and cataract progression (3).
1.2.5.2 Nanoparticles
Nanoparticles have been studied extensively as drug carriers in ocular pharmaceuticals
(44-47). They can be made from biodegradable, polymeric materials in which the drug can be
8
dissolved, entrapped, or adsorbed (48). The major benefits for implementing nanoparticles for
ocular drug delivery are:
1. Ease of administration via injection due to size of particle.
2. Smaller particles are tolerated well in the eye.
3. Particles at the nanometer range have shown increased solubility, surface area,
and drug dissolution.
4. Nanoparticles can be manipulated by polymer’s weight and hydrophilicity to
allow sustained drug release.
5. Particles approximately 200 nm in size can be localized in RPE cells.
Recent studies have shown that after intravitreal injection, a majority of nanoparticles
were localized at the RPE within 6 hours and cytoplasmic concentrations of the NP remained
elevated for as long as 4 months in rats (49).
PLGA is an FDA-approved polymer that has been studied for biocompatibility and
toxicity. The rate of degradation can be manipulated by the polymer’s molecular weight,
hydrophilicity, and ratio of lactide to glycolide in order to extend the release time of drugs (50).
Polyethylene glycol (PEG) also has a slow clearance from blood, allowing increased drug
release. PEG reduces uptake by reticulo-endothelial system in blood compared with unmodified
PLGA RES system clearance (51-53). When bevacizumab, an anti-VEGF antibody, was
incorporated in PLGA-PEG, it showed sustained release for up to 60 days (54). In another study,
when corticosteroid triamcinolone acetonide was encapsulated by PLGA, inflammation
associated with ocular diseases was reduced. The drug/polymer ratio was shown to affect the
entrapment efficiency and release profiles (47).
1.2.5.3 Thermoreversible gels
Thermoreversible hydrogels are a subtype of in situ gels that can be administered as a
solution and undergo gelation with specific stimuli (4, 5). Specifically, thermoreversible gels are
biodegradable, water-soluble polymers that undergo phase transition upon temperature elevation
(36, 55). The gel can be loaded with bioactive macromolecules and pharmacological agents
irrespective of their solubility properties. Since the gel forms quickly in vivo, it can be used to
achieve localized and sustained release. They are attractive due to their ease of administration
9
and improved bioavailability. Studies have shown synthesis of an ABA-type block copolymer,
poly(ethylene glycol)-poly(serinol hexamethylene urethane), to release bevacizumab (55, 56).
The drug release profile demonstrated a sustained release over 17 weeks in vitro. Such therapy
treatment could potentially reduce intravitreal injection frequency (55). Additional studies of
thermo gels based on PLGA and PEG were able to deliver 45kDa protein across the sclera to the
retina for up to 14 days (42). An alternative formulation includes Pluronic F 127 as the
thermoreversible polymer and methylcellulose as a release-controlling agent. This formulation
has been used to deliver nonsteroidal anti-inflammatory drugs for conjunctivitis (57) as well as
selective inhibitors for glaucoma treatment (58).
10
1.3 Hypothesis and Specific Aims
I hypothesize that sustained-release of corticosteroid-nanoparticles incorporated in
thermoreversible gels can improve the therapeutic profiles of corticosteroids for choroidal
neovascularization in wet age-related macular degeneration by reducing the frequency of
intravitreal injections. The hypothesis is tested by the following three specific aims:
1. Preparation and characterization of corticosteroid-encapsulated nanoparticles
incorporated into thermoreversible gels. Nanoparticles including corticosteroid are
prepared by an emulsion solvent evaporation technique and incorporated into a
thermoreversible gel. The drug delivery system is characterized by obtaining size, shape,
drug encapsulation, gelation properties and in vitro sustained release profiles.
2. Examination of the effects of the corticosteroids, nanoparticles, and thermoreversible gel
on cytotoxicity, VEGF expression, and cellular uptake in ARPE-19 cells.
3. Determination of the effect of corticosteroid-nanoparticles incorporated in
thermoreversible gel on chemically induced choroidal neovascularization in mice. The
effects of the drug delivery system on the size of choroidal neovascularization area in a
mouse model are examined.
This study examines the potential of a sustained drug delivery system to reduce the frequency
of intravitreal injections in the treatment of choroidal neovascularization. The drug delivery
system utilizes a dual approach with a PLGA-PEG-PLGA triblock thermoreversible polymer
to suspend corticosteroid nanoparticles. The thermoreversible polymer will hold the drug-
encapsulated nanoparticles at the site of administration and allow for controlled release of the
corticosteroids over a longer duration. This work can help lead to the development of more
effective therapies for age-related macular degeneration.
11
Chapter 2. Triamcinolone Acetonide Nanoparticles Incorporated in
Thermoreversible Gels for Age-Related Macular Degeneration
Anjali Hirani1,2, Aditya Grover1, Yong W. Lee2, Yashwant Pathak1, Vijaykumar Sutariya1*
1 Department of Pharmaceutical Sciences, USF College of Pharmacy, University of South
Florida, Tampa, FL 33612 2 School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University,
Blacksburg, VA 24061
Manuscript published in Pharmaceutical Development and Technology 2014 Sep 26:1-7. [Epub
ahead of print]
2.1. Abstract
Age-related macular degeneration (AMD) is one of the leading causes of blindness in the US
affecting millions yearly. It is characterized by intraocular neovascularization, inflammation, and
retinal damage which can be ameliorated through intraocular injections of glucocorticoids.
However, the complications that arise from repetitive injections as well as the difficulty posed by
targeting the posterior segment of the eye make this interesting territory for the development of
novel drug delivery systems. In the present study, we described the development of a drug
delivery system composed of triamcinolone acetonide-encapsulated PEGylated PLGA
nanoparticles incorporated into PLGA-PEG-PLGA thermoreversible gel and its use against
VEGF expression characteristic of AMD. We found that the nanoparticles with mean size of
208±1.0 nm showed uniform size distribution and exhibited sustained release of the drug. We
also demonstrated that the polymer can be injected as a solution and transition to a gel phase
based on the biological temperature of the eye. Additionally, the proposed drug delivery system
was non-cytotoxic to ARPE-19 cells and significantly reduced VEGF expression by 43.5 ± 3.9%
as compared to a 1.53 ± 11.1% reduction with triamcinolone. These results suggest the proposed
12
drug delivery system will contribute to the development of novel therapeutic strategies for age-
related macular degeneration.
2.2. Introduction
Triamcinolone acetate (TA), a synthetically-modified glucocorticoid, is utilized for its
anti-inflammatory and immunomodulatory effects against a number of diseases (59). TA is
commonly used in conjunction with other drugs and acts by binding steroid receptors in cells and
subsequently inducing or repressing target genes. This leads to an inhibition of inflammatory
processes, such as edema and vascular permeability (59, 60). TA and similar glucocorticoids also
act on vascular endothelial growth factor (VEGF) by inhibiting its secretion and inhibiting
cytokine production (61). Such drugs can also inhibit basic fibroblast growth factor (bFGF)
along with decreasing the VEGF levels characteristic of neovascularization (62). TA was shown
to inhibit laser-induced choroidal neovascularization in rats as well as improve visual acuity
when injected intravitreally (63, 64). However, high doses of TA and similar steroids may lead to
adverse effects such as increased intraocular pressure and the formation of cataracts (65). TA is
clinically used intravitreally against neovascularization in age-related macular degeneration
(AMD) (59).
AMD, a progressive, inflammatory eye disease, is one of the leading causes of blindness
(66). It was estimated that 36.8 million people suffered from some sort of vision loss due to eye
diseases in the United States in 2010 (32). AMD affects the posterior segment of the eye through
damage to retinal pigment epithelium (RPE) cells and leads to a loss of central, focused vision
through the abnormal growth of blood vessels damaging the macula of the retina, the area
responsible for fine vision (67, 68). The presence of intraocular debris may induce an
inflammatory response which may cause further damage to the retina through the induction of a
sustained immune response (69). Advanced AMD is characterized by choroidal
neovascularization (CNV), the growth of abnormal blood vessels beneath the RPE or between
the RPE and retina, accompanied by fluid and blood rupturing Bruch’s membrane into the
subretinal space and leading to retinal irregularities (70). Physical ocular barriers and routes of
treatment pose limitations to therapeutic intervention in treating AMD (71).
Anti-angiogenic therapy is useful in slowing the progression of AMD due to the
neovascularization characteristic of the disease. Vascular endothelial growth factor A (VEGF-A)
13
is the most potent promoter of angiogenesis and vascular permeability and its role in the
pathogenesis of neovascular AMD is well recognized (72, 73). VEGF-A levels are elevated in
human CNV and its vitreous levels have been reported to be increased when compared to healthy
controls (15). VEGF is a potential pharmaceutical target; elevated VEGF levels can increase
inflammation via inducing inflammatory mediators like intercellular adhesion molecule-1
(ICAM-1) and subsequently lead to the breakdown of the blood retinal barrier (BRB) (74, 75).
The anatomy of the eye is a challenging part of the body for drug delivery. The posterior
segment of the eye consists of the retina, vitreous, and choroid. Topical treatment can easily
access the anterior portion of the eye; however, multiple physical barriers and clearance
mechanisms prevent easy access to the posterior segment of the eye. The topical route is the
most favored and convenient for drug delivery, but nasolacrimal drainage and systemic
absorption results in a poor drug bioavailability (76). A model of transient diffusion has shown
that less than 5% of a lipophilic drug and 0.5% of a hydrophilic drug reach the anterior chamber
(74). The bioavailability of the drug further decreases across the sclera, choroid, and RPE (75).
Drug permeability through the sclera is reduced with cationic and lipophilic solutes and RPE
have tight intercellular junctions to prevent the permeation of hydrophilic molecules (75).
Furthermore, the lymphatic system, blood vessels, and active transporters all work to clear drugs
administered through transscleral routes. Drug delivery through systemic administration requires
high doses to obtain a therapeutic concentration in the posterior segment of the eye due to the
tight barriers in RPE. Intravitreal injections circumvent the physiological barriers and maintain
therapeutic doses without damaging bystander tissue; however, frequent injections can lead to
complications like retinal detachment, increase in ocular pressure, and hemorrhage (40). Given
the presence of these physiological barriers, the development of therapies that efficiently deliver
drugs and extend drug release to the posterior segment of the eye would be beneficial to the
progression of ocular disease treatment.
Although current therapies exist to slow the progression of AMD, alternative drug
delivery systems (DDS) are needed to enhance the therapeutic profiles of these drugs.
Nanoparticles (NP) have been studied as drug carriers in ocular pharmaceuticals (77-80). They
can be made from biodegradable, polymeric materials in which drugs can be dissolved,
entrapped, or adsorbed (81). The major benefits for implementing NPs for ocular drug delivery
are: 1) Ease of administration via injection due to the size of the particle, 2) smaller particles are
14
well tolerated in the eye, 3) particles of the nanometer range have shown increased solubility,
surface area, and drug dissolution, 4) NPs can be manipulated by polymer’s weight and
hydrophilicity to allow sustained drug release, and 5) particles approximately 200 nm in size can
be localized in RPE cells. In addition, recent studies have shown that after intravitreal injections,
a majority of NPs were localized at the RPE within 6 hours and cytoplasmic concentrations of
the NP remained elevated for as long as 4 months (82).
Poly(lactide-co-glycolic acid) (PLGA) is an FDA-approved polymer that has been
studied due to its biocompatibility and toxicity. The rate of degradation can be manipulated by
the polymer’s molecular weight, hydrophilicity and ratio of lactide to glycolide to extend the
release time of associated drugs (83). PLGA NPs have been shown to have controlled drug
release, low cytotoxicity, and few side effects (84, 85). Polyethylene glycol (PEG) has a slow
clearance from the blood, allowing an increased drug release and reducing PLGA NP uptake by
the reticulo-endothelial system (RES) when chemically conjugated to the PLGA vector as
compared to non-conjugated PLGA (84-86). PEGylated PLGA NPs of bevacizumab, an anti-
VEGF antibody, showed sustained release of the drug over 60 days (87). In another study, TA
encapsulated in PLGA NPs revealed a decrease in inflammation associated with ocular diseases
(80).
Thermoreversible hydrogels (TR gels) are a subtype of in situ gels that can be
administered as a solution and undergo gelation with specific stimuli (88, 89). Specifically, TR
gels are biodegradable, water soluble polymers that undergo phase transitions upon temperature
elevation (76, 90). The gel can be loaded with bioactive macromolecules and pharmacological
agents irrespective of their solubility properties. Localized and sustained release can be achieved
due to the gel’s quick formation in vivo. Their improved bioavailability and ease of
administration make them an attractive DDS. Studies have shown the synthesis of an ABA-type
block copolymer, poly(ethylene glycol)-poly(serinol hexamethylene urethane), to release
bevacizumab. The in vitro drug release profile achieved a longer therapeutic window over 17
weeks. Such treatments could potentially reduce the frequency of intravitreal injections (90). An
alternative formulation includes Pluronic F 127 as the thermoreversible polymer and methyl
cellulose as a release controlling agent. This formulation has been used to deliver nonsteroidal
anti-inflammatory drugs for conjunctivitis as well as selective inhibitors for glaucoma (91, 92).
15
The present study described the preparation of TA encapsulated PLGA-PEG NPs
incorporated into TR gel to improve the therapeutic profile of TA in AMD. We showed that the
NP size is appropriate for intracellular uptake, the NPs and TR gel demonstrate a sustained
release of the drug over 10 days, the NP and TR gel vectors are nontoxic in ARPE-19 cells, and
the NP and TR gel DDS is able to reduce VEGF levels in ARPE-19 cells. These results suggest
that the proposed DDS has the potential to significantly improve current therapies against AMD.
2.3. Materials and Methods
2.3.1. Materials
Poly(lactic-co-glycolic acid) (PLGA) conjugated with polyethylene glycol (PEG) (PEG-PLGA)
(5050 DLG, mPEG 5000, 5wt% PEG) was purchased from Lakeshore Biomaterials
(Birmingham, AL). Triamcinolone acetonide was purchased from Alfa Aesar (Ward Hill, MA).
Poly(lactide-co-glycolide)-b-Poly(ethylene glycol)-b-Poly(lactide-co-glycolide) [PLGA-PEG-
PLGA (Mn ~ 1,100:1,000:1,100 Da, 3:1 LA:GA) (25% PEG)] thermogelling polymer was
purchased from Polyscitech (West Lafayette, IN). Phosphate buffered saline (PBS) solution was
purchased from Mediatech, Inc (Manassas, VA). Thiazolyl blue tetrazolium bromide (MTT
reagent), acetone and methanol were purchased from Sigma Aldrich (St. Louis, MO). All other
chemicals used in the study were of analytical grade and were used without any further
purification unless specified.
2.3.2. Nanoparticle synthesis
TA NPs were prepared using a previously reported nanoprecipitation method (80, 84). Briefly,
22 mg of TA and 110 mg of PEGylated PLGA were dissolved in 2 mL acetone (organic phase)
and the resulting solution was added dropwise to 20 mL of deionized H2O (40o C) spinning at
300 rpm overnight to allow the full evaporation of the organic phase and the formation of the NP
suspension. The NPs were separated by centrifugation at 3,000 rpm for 10 minutes and
resuspended in fresh deionized H2O.
16
2.3.3. Nanoparticle characterization
The NP parameters were studied by measuring the particle size and polydispersity index (PDI).
The effect of loading TA into the NPs on these parameters was studied by comparing TA NPs to
blank NPs. Particle size and PDI were measured using the Dynamic Pro plate reader (Wyatt
Technology Corporation, Santa Barbara, CA). The NP samples were diluted 1:5 in deionized
water to fit instrument specifications.
2.3.4. Drug encapsulation efficiency
Drug encapsulation efficiency was determined by using a previously reported method (84). 1 mL
of the NP solution was centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed
and was replaced with 1 mL of methanol and stored at 4o C overnight. The supernatant of the
NP/methanol solution was diluted 1:5 times and measured by UV spectroscopy at a wavelength
of 240 nm (λmax). The supernatant was analyzed and compared to a series of standard dilutions of
TA in methanol (r2=0.99764). Encapsulation efficiency was determined using the following
equation:
% 𝐸𝑛𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 𝐴𝑐𝑡𝑢𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑚𝑜𝑢𝑛𝑡
𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑚𝑜𝑢𝑛𝑡× 100
2.3.5. Preparation of thermoreversible gels
TR gels were prepared using the cold method (93). To prepare 20% w/v gel, 100 mg of PLGA-
PEG-PLGA gel was solubilized in deionized H2O overnight at 4o C. Different percentages of the
gel (10, 15, 20, 25, 30% w/v) were screened for the most optimal gelation temperature.
2.3.6. Determination of gelation temperature
Gelation temperature was detected by visual inspection using a previously published method (89,
94). Aqueous solutions of the gel (10, 15, 20, 25, 30% w/v) were prepared in distilled deionized
H2O and 500 μL of each solution was heated from 10o C to 60o C. At each 1o C interval, the
tubes were inverted to investigate flow properties. Solutions were considered to be in the gel
state if no flow was observed following tube inversion.
17
2.3.7. In vitro release
NPs: The rate of TA release from the NPs was measured by adapting a previously reported
method (95). Dialysis membrane tubing, MWCO 10,000 Da. (Spectrum Laboratories, Rancho
Dominguez, CA) was soaked in deionized H2O overnight. 1 mL of the NP suspension was added
to the dialysis membrane tubing after being briefly sonicated, and were placed into 100 mL of
PBS covered and stirring at 100 rpm at 37o C. 1 mL samples were removed at predetermined
intervals over 10 days and replaced with fresh PBS. Samples were analyzed using UV
spectroscopy at a wavelength of 240 nm (λmax) and compared to standard dilutions of TA in PBS
(r2=0.989) to determine the percentage of drug released over 10 days.
TR gel: The rate of TA release from the TR gel was measured by dispersing TA NPs in 200 µL
of 20% w/v polymer solution and allowed to gel by placing in incubator at 37°C for 5 minutes.
After gelling, release was initiated by adding 2.5 mL PBS. 1 mL samples were removed at
predetermined intervals over 10 days and replaced with fresh PBS (94).
2.3.8. Cell culture
Human retinal pigment epithelium, ARPE-19, cells (ATCC # CRL-2302) were grown in 1:1
Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium (Mediatech, Inc., Manassas, VA)
with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin (Sigma Aldrich, St. Louis,
MO). The cultures were maintained in a humid environment at 5% CO2 and 37o C.
2.3.9. Cytotoxicity
The cytotoxicity of the DDS was assessed in ARPE-19 cells by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide salt (MTT) assay. Briefly, cells were seeded in 24-well plates
at a density of 5 x 104 cells/mL for 24 hours to achieve confluency before treatment. Cells were
exposed to free TA, TA NPs, and TA NP-TR gel at varying concentrations (0, 1, 10, 100 μM) for
24 hours after which the MTT assay was performed. Cell culture media were aspirated and 500
μL of MTT reagent solution (1 mg/mL) was added to each well. Cells were incubated at 37o C
and 5% CO2 for 4 hours, after which the MTT reagent solution was aspirated and 1 mL of
DMSO was added to each well. Plates were allowed to shake gently for 10 minutes before being
read by Synergy H4 plate reader (Biotek Industries, Inc., Winooski, VT) at absorbance of 570
nm.
18
2.3.10. VEGF secretion
ARPE-19 cells were seeded onto 24-well plates at 5 x 104 cells/mL and allowed to grow until
confluence. On the day of study, cell culture media were replaced with 1% FBS experimental
media and allowed to remain in quiescence for 12 hours. The cellular monolayers were incubated
with varying treatment concentrations of free TA solution (1, 10, 100 μM), TA NPs (10, 100
μM), and TA NP-TR gel (10, 100 μM). Culture media were collected at 12 and 72 hours.
Secreted VEGF in collected culture media was quantified by the ELISA method (Human
VEGFA ELISA kit, Thermo Scientific, Waltham, MA). The cell protein content was assayed
using the BCA protein assay kit after lysing the cells and VEGF secretion was normalized to
total protein. Samples were read by using the Synergy H4 plate reader (Biotek Industries, Inc.,
Winooski, VT) with absorbance at 450 nm minus absorbance at 550 nm.
2.3.11. Statistical analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., San Diego,
CA). Comparisons of the effect of free TA solution, TA NPs, and TA NP-TR gels on cell
viability and VEGF expression were assessed using paired t-tests with a significance level (p) of
0.05. All experiments were carried out in triplicates (n=3) and shown as mean values ± SD.
2.4. Results
2.4.1. Nanoparticle characterization
Dynamic Pro plate reader was used to determine the size and PDI of the blank and TA-loaded
NPs. The TA NPs were larger in size than the blank NPs. PDI data revealed that all NP
formulations had a narrow average size distribution (Table 2.1).
Table 2.1: NP characterization data carried out in triplicates (n=3) and represented as the mean value ± SD.
NP Type: Particle size (nm): PDI:
Blank NP 125.10 ± 43.89 0.014 ± 0.004
TA NP 208.00 ± 1.00 0.005 ± 0.001
19
2.4.2. Drug encapsulation efficiency
UV spectroscopy was used to determine the encapsulation efficiency of TA by comparing the
absorbance in methanol to standard dilutions of TA in methanol (r2=0.99764) at 240 nm (λmax). It
was found that 42.4 ± 0.09% of the TA added during formulation was taken up by the NPs.
2.4.3. Determination of gelation temperature
Phase transition studies revealed that the 15% w/v aqueous solution of the polymer converts to
the gel phase at 40o C and remains in a gel state until 47o C. The gel phase is broader with higher
% w/v solutions. The 20% w/v gel solution demonstrated an optimal transition within
physiological conditions and was employed for future studies. Figure 2.1 shows the temperature
at which each gel formulation transitioned from the solution state to gel state. Figure 2.2 gives a
visual depiction of the phase change from a solution at 4o C to a gel at 37o C with the 20% w/v
polymer.
20
Figure 2.1: Temperature-dependent phase transitions in PLGA-PEG-PLGA thermoreversible gels based on w/v concentration of gel in aqueous solution. The 20% w/v thermoreversible gel was chosen for further studies because its phase transition occurs over physiologically relevant temperatures.
21
Figure 2.2: Pictorial representation of the temperature-dependent phase change exhibited by the 20% w/v PLGA-PEG-PLGA thermoreversible gel. The gel solution exists in the liquid state at 4o C and transitions into a gel state at 37o C.
2.4.4. In vitro release
The release of TA from NPs and TR Gel was investigated in 100 mL of PBS and 37o C. Figure
2.3 shows the cumulative release profile of the TA NP formulation and TA NP Gel as compared
to equal concentrations of the free TA solution. At 48 hours, free TA was fully released while the
TA NPs released 48.9 ± 7.0% of encapsulated drug and the TA NP Gel released 23.24 ± 6.7% of
drug. At 168 hours, the cumulative release of TA from the NP formulations was 94.17 ± 20.8%.
There is a lack of initial burst release of drug from the NPs. TR Gel had released 31.49 ± 5.1% of
drug at 10 days.
22
Figure 2.3: In vitro release data of the TA NP and TA NP Gel as compared to equal concentrations of the TA drug. Samples were analyzed by UV spectroscopy (240 nm, λmax) at predetermined intervals over a 10-day period (n=3, mean value ± SD).
2.4.5. Cytotoxicity
The cytotoxicity of the DDS was investigated by MTT assay in the ARPE-19 cells to determine
any possible harm related to its use. Figure 2.4 compares the cytotoxicity data of the free TA
with equal concentrations (10 µM) of the different TA DDS’s described in the present study: The
blank NPs, TA NPs, the TA in 20% w/v TR gel, and the TA NP in 20% w/v TR gel. The free TA
induced significant cell death in ARPE-19 cells as compared to the untreated control cells. All
other treatments were not significantly cytotoxic in ARPE-19 cells.
23
Figure 2.4: MTT cytotoxicity data in ARPE-19 cells of equal treatments (10 μM) of the following: Blank NP, TA free drug, TA NPs, TA drug in 20% w/v TR gel, and TA NPs in 20% w/v TR gel as compared to untreated control cells. Experiments were carried out in statistical triplicates (n=3) and quantified by absorbance reading at 570 nm (Synergy H4 Plate reader, Biotek Industries Inc.). Data is represented as mean number of viable cells ± SD. Statistical tests were carried out using paired t-test (p≤0.05).
2.4.6. VEGF secretion
The effect of the different TA NP formulations on VEGF secretion was studied in ARPE-19
cells. Cells were treated with different concentrations of free TA, TA NPs, TA in 20% w/v TR
gel, and TA NP in 20% w/v TR gel, for 12 and 72 hours. Figure 2.5 compares the time-
dependent suppression of VEGF expression among blank NPs, free TA, and TA NPs (100 µM)
at 12 and 72 hours. The blank NPs did not reduce VEGF expression throughout the time periods
tested. The free TA significantly reduced VEGF secretion in 12 hours, but TA NPs were not able
to do it; however, at 72 hours, free TA was unable to significantly reduce VEGF secretion but
TA NPs were able to reduce VEGF secretion. Figure 2.6 compares the effect of different DDS’s
24
(10 µM) on VEGF expression at 72 hours: free TA, TA NPs, TA in 20% w/v TR gel, and TA
NPs in 20% w/v TR gel. The TA NPs, TA in 20% w/v TR gel, and TA NPs in 20% w/v TR gel
all significantly reduced VEGF expression after 72 hours, but the free TA alone had no
significant effect.
Figure 2.5: Time-dependent inhibition of VEGF secretion in ARPE-19 cells through equal concentration treatments (100 μM) of blank NPs, TA free drug, and TA NPs at 12 and 72 hours. Experiments were carried out using the ELISA method (Human VEGFA ELISA kit, Thermo Scientific) in statistical triplicates (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD.
25
Figure 2.6: Comparative suppression of VEGF secretion in ARPE-19 cells through equal concentration treatments (10 μM) of TA free drug, TA NPs, TA drug in 20% w/v TR gel, and TA NPs in 20% w/v TR gel at 72 hours. Experiments were carried out using the ELISA method (Human VEGFA ELISA kit, Thermo Scientific) in statistical triplicates (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD.
2.5. Discussion
The emulsion solvent evaporation process was used in the formulation of the polymeric
TA NP suspension. Multiple formulations revealed consistent reproducibility with respect to
particle size and encapsulation efficiency (data not shown). Particle size data showed that the
average size of the NPs increases with drug loaded into the NPs as compared to the blank NPs
(180-208 nm). Each of the NP formulations exhibited a unimodal size distribution, suggesting a
Gaussian distribution with respect to NP size in each formulation. Particle size, PDI, and
encapsulation efficiency data were corroborated with previously published studies (80, 96-98).
26
The release of TA from the TA NPs and TR gel was measured in 37o C PBS over 10 days
as compared to the release of the free TA. TA remains stable in solution as demonstrated by
release data previously reported (80, 99, 100). The free TA was completely released from the
dialysis membrane in the first 48 hours; however, only approximately 48.9 ± 7.0% of the drug
was released from each of the NP formulations in as much time. Almost all of the drug was
released from the NPs by the end of the 10 day period. The lack of initial burst release suggests
that the drug was well encapsulated by the PLGA NP during formulation and none of the drug
exists on the surface of the NP. The NP formulation exhibited a sustained release over the 10 day
period tested. The release of entrapped drug from a polymer matrix is believed to occur in two
stages: The early stage of drug release occurs through diffusion in the polymer matrix while the
latter phases occur through a combination of diffusion and polymer degradation (101). 31.49 ±
5.1% of the drug was released from the TR gel at 10 days. Similar results are reported in the
literature where TR gels based on PLGA and PEG were able to deliver a 45 kDa protein across
the sclera to the retina for up to 14 days (102).
The gelation temperatures of different % w/v formulations of the PLGA-PEG-PLGA TR
gel were investigated. It was found that an increasing % w/v of the TR gel solution broadened
the temperature range over which the TR gel changes phases from solution to gel to solution
(sol-gel-sol). The temperatures over which the 20% w/v TR gel phase transitioned from sol-gel-
sol were best in regards to physiological conditions. The 20% w/v TR gel transitions from
solution to gel at approximately 32o C, which is the optimum temperature for its easy intraocular
injection and phase transition into the gel phase at physiological temperatures. The incorporation
of a 10 μM treatment of TA NP formulation into the 20% w/v gel did not produce any cytotoxic
effects on ARPE-19 cells, suggesting its safe use in in vivo settings.
NP cytotoxicity studies in ARPE-19 cells showed that the PLGA NP vector is not
cytotoxic although treatments with 10 μM free TA are cytotoxic under similar conditions. It was
also found that not only is the PLGA vector safe for use in ARPE-19 cells but also that the TA
NP formulation and TR gel DDS (with incorporated TA NPs) are not significantly cytotoxic.
Concentrations of TA needed to can inhibit VEGF secretion have been previously reported
(103). Consistent with those values, our studies have shown the TA NPs and TR DDS
formulations were able to significantly reduce VEGF expression in ARPE-19 cells at 10 and 100
μM over 72 hours more effectively than the free TA at equal concentrations. The free TA is able
27
to significantly reduce VEGF expression at the 100 µM concentration in 12 hours and similar
concentrations take the TA NPs 72 hours to reduce VEGF expression, which may be due to the
extended release properties of the TA NPs.
2.6. Conclusion
The present study describes the potential use of a novel DDS for the amelioration of the
CNV exhibited in cases of AMD. The DDS consists of two parts: TA encapsulated NPs that are
incorporated into 20% w/v TR gels. The TA NPs are spherical and the shape and sizes are
compatible in the cells. The NPs exhibited an extended and sustained release of TA as is evident
from in vitro release data. Furthermore, the TA NPs and NP-incorporated 20% w/v TR gel did
not exhibit cytotoxicity in ARPE-19 cells in concentrations that are shown to significantly reduce
ARPE-19 cell viability by free TA alone under the same conditions. The TA NP and NP-
incorporated 20% w/v TR gel were able to significantly reduce the expression of VEGF in
ARPE-19 cells over 72 hours at concentrations less than those required by free TA alone (10 µM
TA-loaded DDS vs. 100 µM free TA). The biocompatibility and therapeutic potential of the
proposed DDS makes it an effective model for further studies investigating the pathology and
treatment of AMD.
Declaration of Interest:
The authors report no declarations of interest.
28
Chapter 3. Efficacy of Loteprednol Etabonate Drug Delivery System
in Suppression of in vitro Retinal Pigment Epithelium Activation
Anjali Hirani1,2, Yong W. Lee2, Yashwant Pathak1, Vijaykumar Sutariya1*
1 Department of Pharmaceutical Sciences, USF College of Pharmacy, University of South
Florida, Tampa, FL 33612 2 School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University,
Blacksburg, VA 24061
Manuscript accepted in Pharmaceutical Nanotechnology.
3.1 Abstract
Choroidal neovascularization (CNV) is the growth of abnormal blood vessels in the choroid
layer of the eye; it is a pathophysiological characteristic of wet age-related macular degeneration
(AMD). Current clinical treatment utilizes frequent intravitreal injections, which can result in
retinal detachment and increased ocular pressure. The purpose of the current study is to develop
a novel drug delivery system of loteprednol etabonate-encapsulated PEGylated PLGA
nanoparticles incorporated into the PLGA-PEG-PLGA thermoreversible gel for treatment of
AMD. The proposed drug delivery system was characterized for drug release, cytotoxicity
studies and vascular endothelial growth factor (VEGF) suppression efficacy studies using ARPE-
19 cells. The nanoparticles showed uniform size distribution with mean size of 168.60 ± 23.18
nm and exhibited sustained drug release. Additionally, the proposed drug delivery system was
non-cytotoxic to ARPE-19 cells and significantly reduced VEGF expression as compared to
loteprednol etabonate solution. These results suggest the proposed drug delivery system can be
29
used for further work in an animal model of experimental AMD with reduced intravitreal
administration frequency.
3.2 Introduction
Loteprednol etabonate (LE), a derivative of prednisolone was the first retrometabolically
designed steroid. It is a site-active corticosteroid designed to transform into an inactive
metabolite after exerting its therapeutic effect. It contains an ester group at the carbon 20 position
instead of a ketone (104-108). Compared to prednisolone acetate, it is less likely to increase
ocular pressure and does not lead to cataract formation. Therefore, it is commonly prepared as a
suspension and used topically for anterior segment disorders such as allergy conjunctivitis,
anterior uveitis, and postoperative inflammation (109, 110).
Physical barriers and routes of drug administration create serious limitations in treating AMD
(71). The eye is extremely challenging for drug delivery. Physical barriers and clearance
mechanisms prevent access to the posterior segment of the eye. The bioavailability of a drug
further decreases across the sclera, choroid, and retinal pigment epithelium (RPE) (75).
Moreover, the lymphatic system, blood vessels, and active transporters all work to clear drugs
administered. As a consequence, most formulations have short residence times within the eye.
Due to these barriers, the development of therapies that prolong ocular residence time and
enhance drug delivery and release to the posterior segment of the eye would be beneficial to the
progression of ocular disease treatment.
Currently, the most efficient method to administer therapeutics for AMD is by intravitreal
injections. The vitreous is gelatinous in nature, which is capable of retaining drug molecules and
also delivering them to nearby structures, such as the RPE or ciliary body. Intravitreal injections
circumvent the physiological barriers and maintain therapeutic doses without damaging
30
bystander tissue; however, frequent injections are necessary and can lead to complications like
retinal detachment, increase in ocular pressure, and hemorrhage (40). Therefore, alternative drug
delivery systems (DDS) are needed to enhance the therapeutic profiles of these drugs.
Nanoparticles (NPs) made from biodegradable, polymeric materials in which drugs can be
dissolved, entrapped, or adsorbed (81) have been studied as drug carriers for effective drug
delivery to the posterior segment in ocular pharmaceuticals (77-80). Recent research has shown
that NPs can localize within the RPE and maintain therapeutic effect for up to 4 months (111-
113). In one study, Bourges et al. have reported distribution of polylactic acid (PLA) NPs loaded
with Rh-6G fluorochromes in RPE cells of the healthy rat after 24 hours. The fluorochrome was
visible for 4 months after the single injection. Another report studied different nanosuspensions
prepared of three insoluble glucocorticoids, such as hydrocortisone, prednisolone, and
dexamethasone, and revealed higher bioavailability and therapeutic effects of the glucocorticoid
action (114). Recently, poly(lactide-co-glycolic acid)-polyethylene glycol (PLGA-PEG) NPs
have been shown to have controlled drug release, low cytotoxicity, and few side effects (84, 85).
Chemical conjugation with PEG reduces clearance from the blood, allowing an increased drug
release and reducing PLGA NPs uptake by the reticulo-endothelial system (RES) (84-86).
PEGylated PLGA NPs of anti-VEGF antibodies have shown sustained release of the drug over
60 days (87). Other PLGA DDS that have been approved for ophthalmic applications include
OzurdexTM; this implant releases dexamethasone intravitreally over 4 to 6 weeks. Results have
shown improvement in inflammation; however surgery is required for implant and removal (3,
43). Alternative PLGA biodegradable implants are being investigated for use in cytomegalovirus
retinitis with the release of ganciclovir, and for proliferative vitreoretinopathy with the release of
all-trans retinoic acid (115).
31
Thermoreversible hydrogels (TR gels) are biodegradable, water soluble polymers that
undergo phase transitions upon temperature changes (76, 90). These polymers can be loaded
with therapeutic agents and can give sustained drug release in vivo. They are effective for use as
DDS due to their improved bioavailability and ease of administration. In vitro drug release
profile of bevacizumab loaded in an ABA-type block copolymer have demonstrated a sustained
drug release over 17 weeks (90). Moreover, PLGA-PEG TR gels have also been used to deliver
proteins to the retina for up to 2 weeks (102).
The present study describes a drug delivery system consisting of loteprednol etabonate
encapsulated PLGA-PEG NPs incorporated into TR gel to improve the therapeutic profile of
loteprednol etabonate in AMD. The proposed DDS has the potential to significantly improve
current therapies against CNV.
3.3 Materials and Methods
3.3.1 Materials
Poly(lactic-co-glycolic acid) (PLGA) conjugated with polyethylene glycol (PEG) (PEG-PLGA)
(5050 DLG, mPEG 5000, 5wt% PEG) was purchased from Lakeshore Biomaterials
(Birmingham, AL). Loteprednol etabonate was purchased from Selleckchem (Houston, TX).
Poly(lactide-co-glycolide)-b-Poly(ethylene glycol)-b-Poly(lactide-co-glycolide) [PLGA-PEG-
PLGA (Mn ~ 1,100:1,000:1,100 Da, 3:1 LA:GA) (25% PEG)] thermogelling polymer was
purchased from Polyscitech (West Lafayette, IN). Phosphate buffered saline (PBS) solution was
purchased from Mediatech, Inc (Manassas, VA). Thiazolyl blue tetrazolium bromide (MTT
reagent), acetone and methanol were purchased from Sigma Aldrich (St. Louis, MO). All other
32
chemicals used in the study were of analytical grade and were used without any further
purification unless specified.
3.3.2 Nanoparticle synthesis
Loteprednol etabonate -loaded NPs (LE NPs) were prepared using a previously reported
nanoprecipitation method (80, 84). 8 mg of LE and 55 mg of PEGylated PLGA were dissolved in
4 mL acetone (organic phase) and added dropwise to 8 mL of deionized H2O spinning at 300
rpm overnight to allow full evaporation of the organic phase and the formation of the NP
suspension. The NPs were separated by centrifugation at 3,000 rpm for 10 minutes and
resuspended in fresh deionized H2O.
3.3.3 Nanoparticle characterization
Parameters of the NPs were studied by measuring the particle size and polydispersity index
(PDI). The effect of loading LE into the NPs on these parameters was studied by comparing LE
NPs to blank NPs. Particle size and PDI were measured using the Dynamic Pro plate reader
(Wyatt Technology Corporation, Santa Barbara, CA). The NPs samples were diluted 1:5 in
deionized water to fit instrument specifications.
3.3.4 Drug entrapment efficiency
Drug entrapment efficiency was determined by using a previously reported method (84). 1 mL of
the NPs solution was centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed and
was replaced with 1 mL of methanol and stored at 4o C overnight. The supernatant of the
NPs/methanol solution was diluted 1:5 and measured by UV spectroscopy at a wavelength of
243 nm (λmax). The supernatant was analyzed and compared to a series of standard dilutions of
33
LE in methanol (r2=0.9981). Encapsulation efficiency was determined using the following
equation:
% 𝐸𝑛𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 𝐴𝑐𝑡𝑢𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑚𝑜𝑢𝑛𝑡
𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑚𝑜𝑢𝑛𝑡× 100
3.3.5 Scanning electron microscopy (SEM)
SEM was utilized to visualize the surface and physical integrity of LE NPs. JOEL JSM-6490LV
(JOEL Industries, Tokyo, Japan) was used to visualize the samples. The samples were diluted
according to instrumental specifications and loaded onto aluminum cylinders coated with an
adhesive carbon polymer. NP formulations were viewed in 15,000x magnification and 5 kv
acceleration voltage was used to visualize the drug loaded NPs.
3.3.6 Preparation of thermoreversible gels
TR gels were prepared using the cold method (93).. Briefly, the 20% w/v gel was prepared by
solubilizing 100 mg of PLGA-PEG-PLGA gel in deionized H2O overnight at 4oC.
3.3.7 In vitro release
LE release rate from the NPs was measured by a previously reported method (95). Dialysis
membrane tubing, MWCO 10,000 Da. (Spectrum Laboratories, Rancho Dominguez, CA), was
soaked in deionized H2O overnight. NPs solutions were sonicated, and 1 mL was added to the
dialysis membrane tubing; tubing was placed into 100 mL of PBS and stirred at 100 rpm at 37o
C. 1 mL samples were removed at predetermined intervals over 7 days and replaced with fresh
PBS. Samples were analyzed using UV spectroscopy at a wavelength of 243 nm (λmax) and
compared to standard dilutions of LE in PBS (r2=0.9915) to determine the percentage of drug
released over 10 days.
34
LE release from the TR gel was measured by allowing 200 µL of LE NPs in 20% w/v polymer
solution to gel by placing in incubator at 37°C for 5 minutes. After gelling, release was initiated
by adding 2.5 mL PBS. 1 mL samples were removed at predetermined intervals over 7 days and
replaced with fresh PBS (94).
3.3.8 Cell culture
Human retinal pigment epithelium, ARPE-19, cells (ATCC # CRL-2302) were grown in 1:1
Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium (Mediatech, Inc., Manassas, VA)
with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin (Sigma Aldrich, St. Louis,
MO). The cultures were maintained in a humid environment at 5% CO2 and 37oC.
3.3.9 Cytotoxicity
Cytotoxicity was assessed in ARPE-19 cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide salt (MTT) assay. Briefly, cells were seeded in 24-well plates at a
density of 5 x 104 cells/mL for 24 hours to achieve confluence before treatment. Cells were
exposed to free LE solution, LE NPs, and LE NPs-TR gel at varying concentrations (0, 1, 10,
100 μM) for 24 hours after which the MTT assay was performed. Cell culture media were
aspirated and 300 μL of MTT reagent solution (1 mg/mL) was added to each well. Cells were
incubated at 37oC and 5% CO2 for 4 hours, after which the MTT reagent solution was aspirated
and 500 μL of DMSO was added to each well. Plates were allowed to shake gently for 10
minutes before being read by Synergy H4 plate reader (Biotek Industries, Inc., Winooski, VT) at
absorbance of 570 nm.
35
3.3.10 Intracellular uptake of coumarin-6-loaded NPs in ARPE-19 cells
Coumarin-6-loaded NPs were prepared by the method outlined above. In lieu of LE, 15 µL of
coumarin-6 (1mg/ml acetone stock solution) was added (85). ARPE-19 cells were seeded at
10,000 cells per well in a four-well chamber slide. The cells were incubated for 24 hours to
achieve 70% confluence. The medium was aspirated and cell monolayer was washed three times
with PBS. 500 µL media containing 50 µg of coumarin-loaded NPs was added to the wells and
incubated for 2 hours. After 2 hours, cells were washed with PBS three times, and treated with
DAPI for nuclear staining and Cell Mask Deep Red for membrane staining. The slide was then
examined by confocal microscopy with a magnification of 60x.
3.3.11 VEGF secretion
ARPE-19 cells were seeded onto 24-well plates at 5 x 104 cells/mL and allowed to grow until
confluence. On the day of study, cell culture media were replaced with 1% FBS experimental
media and allowed to remain in quiescence for 12 hours. The cellular monolayers were incubated
with varying treatment concentrations of free LE solution (1, 10 μM), LE NPs (10 μM), and LE
NPs-TR gel (10 μM). Culture media were collected at 12 and 72 hours. Secreted VEGF in
collected culture media was quantified by the ELISA method (Human VEGFA ELISA kit,
Thermo Scientific, Waltham, MA). The cell protein content was assayed using the BCA protein
assay kit after lysing the cells and VEGF secretion was normalized to total protein. Samples were
read by using the Synergy H4 plate reader (Biotek Industries, Inc., Winooski, VT) with
absorbance at 450 nm minus absorbance at 550 nm.
36
3.3.12 Statistical analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., San Diego,
CA). Comparisons of the effect of free LE solution, LE NPs, and LE NPs-TR gels on cell
viability and VEGF expression were assessed using paired t-tests with a significance level (p
value) of 0.05. All experiments were carried out in triplicates (n=3) and shown as mean values ±
SD.
3.4 Results
3.4.1 Nanoparticle characterization
Dynamic Pro plate reader was used to determine the size and PDI of the blank and LE -loaded
NPs. The LE NPs were larger in size than the blank NPs. PDI data revealed that all NPs
formulations had a normal size distribution (Table 3.1) with PDI value less than 1.
Table 3.1: Nanoparticle (NP) characterization data of particle size and polydispersity index (PDI) carried out in triplicates (n=3) and represented as the mean value ± SD.
NP Type: Particle size (nm): PDI:
Blank NPs 125.10 ± 43.89 0.014 ± 0.004
LE NPs 168.00 ± 23.18 0.0142 ± 0.0023
3.4.2 Drug entrapment efficiency
UV spectroscopy was used to determine the entrapment efficiency of LE by comparing the
absorbance in methanol to standard dilutions of LE in methanol (r2=0.9981) at 243 nm (λmax).
The LE NPs formulation showed an entrapment efficiency of 82.6 ± 0.01% which was
satisfactory.
37
3.4.3 Scanning electron microscopy (SEM) analysis of NPs formulations
SEM was used to visualize the morphology of LE NPs. Surface analysis of NPs formulations
showed that physical integrity was maintained by all samples (Figure 3.1). Size and size
distribution of the NPs formulation visualized through SEM corroborated with data obtained by
DLS.
Figure 3.1: SEM visualization of loteprednol etabonate-loaded nanoparticles shows round morphology. Samples were diluted 1:10 and visualized by SEM. Samples were read at 15,000x magnification and 5kV acceleration voltage.
3.4.4 In vitro release
The release of LE from NPs and TR gel was conducted in vitro in a beaker with 100 mL of PBS
at 37oC. Figure 3.2 shows the cumulative release profile of the Lot NPs formulation compared to
the LE NPs TR gel. At 72 hours, the LE NPs formulation had released 88% of the encapsulated
38
drug while the LE NPs TR gel formulation had released 5% of drug. At 168 hours, the TR gel
had released 10% of drug.
Figure 3.2: In vitro release data of the loteprednol etabonate nanoparticles (LE NP) compared to LE NP gel. Samples were analyzed by UV spectroscopy (243 nm, λmax) at predetermined intervals over a 7-day period (n=3, mean value ± SD).
3.4.5 Cytotoxicity
The cytotoxicity of the DDS was investigated by MTT assay in the ARPE-19 cells to determine
any possible harm related to its use. Figure 3.3 compares the cytotoxic effects of free LE with
equal concentrations of the LE NPs and LE NPs in 20% w/v TR gel. Free LE had no impact on
cell viability in ARPE-19 cells as compared to the untreated control cells. All other treatments
were not significantly cytotoxic in ARPE-19 cells.
39
Figure 3.3: MTT cytotoxicity data in ARPE-19 cells of increasing concentrations of loteprednol etabonate (LE) free drug, LE NPs, and LE NPs in 20% w/v TR gel as compared to untreated control cells. Experiments were carried out in triplicates (n=3) and quantified by absorbance reading at 570 nm. Data is represented as mean ± SD.
3.4.6 Intracellular uptake of coumarin-6-loaded NPs
Figure 3.4 shows uptake of coumarin 6-loaded NPs within 2 hours. From the images, blue
fluorescence from the nuclei was observed in the first panel due to DAPI labeling. Green
fluorescence of the coumarin-6-loaded NPs was observed in the cytoplasm (second panel) and
the cell membrane was stained with Cell Mask Deep Red in the third panel. The uptake of the
40
NPs by the cells is likely to have occurred via endocytosis. Untreated cells did not show green
fluorescence (data not shown).
Figure 3.4: Cellular uptake of coumarin-6-loaded NPs in ARPE-19 cells. Nuclei were stained with DAPI visible in first panel. The uptake of coumarin-6-loaded NPs is depicted in second panel. Membrane staining with Cell Mask Deep Red is shown in the third panel. The final panel displays the overlaying images. Magnification of 60x.
3.4.7 VEGF secretion study
The effect of the LE DDS on VEGF secretion was studied in ARPE-19 cells. Cells were treated
with different concentrations of free LE solution, LE NPs, and LE NPs in 20% w/v TR gel, for
12 and 72 hours. Figure 3.5 compares the suppression of VEGF expression between free LE
solution, LE NPs and the LE NPs TR gel at 12 hours. Free LE solution at 1 µM and 10 µM
showed reduction in VEGF expression compared to media control. LE NP at 10 µM was used for
the VEGF expression study due to slower release characteristic of the NPs. Moreover, our
preliminary data have shown better VEGF expression reduction at this concentration. LE NPs at
10 µM slightly reduced VEGF secretion while NPs TR Gel at 10 µM did not reduce VEGF
secretion significantly in 12 hours compared to the control (p value <0.05) as concluded by
paired t-test. However, the paired t-test results indicated that both NPs and NPs TR Gel at 10
µM significantly reduced VEGF secretion in 72 hours (p value <0.05; figure 3.6). This may be
due to controlled release of the drug from NPs polymer matrix and NPs TR Gel. The VEGF
41
expression for NPs and NPs TR Gel at 10 µM was reduced to 90.99 ±1.29 % and 96.24 ± 1.91 %
respectively. The % reduction VEGF expression of NPs TR Gel was observed as lower than NPs
due to further delayed release of the drug from the gel matrix compared to NPs alone.
Figure 3.5: Comparative suppression of VEGF secretion in ARPE-19 cells through increasing concentrations (1, 10, μM) of loteprednol etabonate (LE) free drug, LE NPs, and LE NP gel at 12 hours. Experiments were carried out using the ELISA method (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD. *p<0.05 vs. Control.
42
Figure 3.6: Comparative suppression of VEGF secretion in ARPE-19 cells through equal concentration treatments (10 μM) of LE free drug, LE NPs, and LE NPs in 20% w/v TR gel at 72 hours. Experiments were carried out using the ELISA method (n=3). Data is represented by amount of VEGF secretion normalized to untreated control levels ± SD. *p<0.05 vs. Control.
3.5 Discussion
Polymeric LE NPs were prepared by the emulsion solvent evaporation method with
reproducible and satisfactory particle size and entrapment efficiency. The particle size of
unloaded NPs was 125 nm while drug loaded NPs was 168 nm. Both NPs formulations showed
a normal size distribution with PDI value less than 1. Particle size, PDI, and entrapment
efficiency data were corroborated with previously published studies (80, 96, 97, 116).
43
The rate of drug release from the NPs and TR gel was measured using dialysis method in
PBS at 37o C over the course of 7 days. At 72 hours, the LE NPs exhibited 88% drug release
while the LE NP Gel had released 5% of drug. The TR gel had released 10% of drug at 168
hours. Both formulations exhibited sustained release pattern; however, the drug release from the
TR gel was further slower due to drug release via polymer degradation. Polymer matrices release
encapsulated drug in two phases: the first phase of drug release occurs via diffusion and the
second phase is by diffusion and polymer degradation (101).
MTT cytotoxicity studies in ARPE-19 cells showed no cytotoxicity after 24-hour
exposure to free LE solution, LE NPs, or LE TR gel. LE was not expected to have any toxic
effects as it was formulated to prevent any physiological side effects. The LE NP and LE NP
incorporated TR gel DDS were observed to be safe to use in ARPE-19 cells. Additionally, the
NPs were localized into the ARPE-19 cells within 2 hours as shown by confocal microscopy
(figure 4). To support these data, recent in vivo studies have shown that NPs were localized
within 6 hours at the retinal pigment epithelium of healthy rat after intravitreal injections (82).
LE NPs and LE NP TR gel were able to significantly reduce VEGF expression in ARPE-19 cells
at 10 μM over 72 hours more effectively than equal concentrations of LE drug solution (p<0.05).
Free LE drug solution significantly reduced VEGF expression within 12 hours compared to
control (p<0.05) while drug loaded NPs and NPs TR Gel exhibit their effect after 72 hours due to
the extended release properties of the NPs and the gel.
44
3.6 Conclusions
The present study describes the potential use of a novel DDS for controlled release of LE
for potential treatment of wet AMD by reducing the frequency of intravitreal injections. The
proposed DDS showed sustained in vitro release and showed no cytotoxicity in in vitro
experiments using ARPE-19 cells. The DDS of LE was able to significantly reduce the
expression of VEGF in ARPE-19 cells over 72 hours compared to LE solution alone. The
biocompatibility, low toxicity and therapeutic potential of the proposed DDS make it an ideal
model for further studies investigating the experimental animal AMD models with reduced
intravitreal injection frequency.
Declaration of Interest:
The authors report no declarations of interest.
Acknowledgements:
Scanning electron microscopy assistance was provided by Amanda Garces and confocal
microscopy assistance was provided by Dr. Byeong "Jake" Cha, Ph.D., Lisa Muma Weitz
Advanced Microscopy and Cell Imaging Core Laboratory (University of South Florida).
45
Chapter 4. The Effect of Corticosteroid-Nanoparticles Incorporated
in a Thermoreversible Gel on Chemically Induced Choroidal
Neovascularization in Mice
4.1 Introduction
Age-related macular degeneration (AMD) is the leading cause of legal blindness in the
developed world. Vision reduction and blindness develop at advanced stages of the disease,
especially in AMD with choroidal neovascularization (CNV) (117, 118). CNV causes
accumulation of exudates in the subretinal space and underneath the retinal pigment epithelium
(RPE). Visual acuity is compromised over time due to damage of the macula.
Due to the neovascularization in wet-AMD, inhibitors of angiogenesis have been widely
studied and implemented clinically to deter the development of abnormal blood vessels.
Corticosteroids, such as triamcinolone acetonide and dexamethasone, are used
extensively for treatment of posterior ocular disorders. This is because of their angiostatic,
antipermeable, and antifibrotic properties (27). They are able to inhibit inflammatory symptoms
by binding steroid receptors in cells and inducing or repressing targeted genes (such as TNF-α,
IL-1β, and IL-6) (28, 119). They can also inhibit expressions of growth factors such as basic
fibroblast growth factor and adhesion molecules such as ICAM-1 and decrease VEGF levels that
are elevated in the process of retinal neovascularization (30, 31).
For treatment of posterior segment ocular diseases such as AMD, the biggest challenge is
route of administration. Intravitreal injections allow for the most direct approach; however, the
chronic nature of the disease requires consistent injections resulting in side-effects such as retinal
detachment and cataract formation (24). Therefore, development of sustained release drug
delivery systems (DDS) can be useful in minimizing the frequent intravitreal injections required
to maintain the therapeutic drug dosage.
The DDS containing corticosteroids has been found to be effective at reducing VEGF
expression in vitro (116). Similarly, they have been shown to reduce VEGF expression and
inhibit VEGF-related permeability in vivo (120, 121). The ability to sustain drug release has also
been shown (116). In this study, we evaluated the efficacy of the DDS (LE DDS: LE NPs in a
TR gel, TA DDS: TA NPs in a TR gel) in reducing CNV in a mouse model.
46
4.2 Materials and Methods
4.2.1 Animals
Male C57BL/6 mice (7-9 weeks old) were purchased from Charles River (Wilmington, MA).
This study was approved by the Institutional Animal Care and Use Committee of the University
of South Florida. They are housed in the USF College of Medicine Vivarium under standard
conditions including 12 by 12 light dark cycle and fed standard food during the whole study.
4.2.2 Study design
To investigate the effects of different corticosteroids and DDS, we divided the animals into six
groups. Group 1 (control) was not treated. Group 2 was CNV-induced but left untreated. Groups
3-6 were CNV-induced and administered intravitreal injections of different treatments (LE
(solution), LE DDS (LE NPs in TR gel), TA (solution), TA DDS (TA NPs in TR gel)).
4.2.3 PEG-induced choroidal neovascularization
Male C57BL/6 mice (7–9 weeks old) were administered subretinal injections of 1 mg of
polyethylene glycol-8 (PEG-8). The vitreous chamber of the mouse eye was decompressed with
a 27-gauge needle by inserting the needle through the conjunctiva and sclera 1 mm behind the
limbus. A Hamilton syringe with a 33-gauge blunt needle was used for injections. Needle
movement was stopped when light resistance was felt. An injection of 2 µl of solutions was
administered (122).
4.2.4 Treatment administration
Treatments were administered one week after CNV induction. Intravitreal injection of 1 µl of
each treatment containing 800 µg/ml of respective drug (LE, LE DDS, TA, or TA DDS) was
administered consecutively in each eye using a dissecting microscope. A 30G ½ needle was
inserted 0.5 mm posterior to the temporal limbus approximately 1.5 mm deep and angled toward
the optic nerve until the needle tip was viewed in the vitreous (24).
47
4.2.5 Preparation of flat mounts and CNV evaluation
At two weeks and four weeks after treatment, the size of the CNV lesions was evaluated. Mice
were anesthetized and perfused with 10 ml of PBS containing 50 mg/ml of fluorescein-labeled
dextran. The eyes were removed and fixed in 4% paraformaldehyde for 2 hours. The cornea and
lens were dissected and the entire retina was removed from the eyecup. 4-8 radial cuts were
made from the edge of the eyecup to the equator and the choroid was flat-mounted in aquamount
(123). Flat mounts were examined at 20x objective on an Olympus FV1000 MPE multiphoton
laser scanning microscope. The percent area of neovascularization was quantified by Fiji
(ImageJ) software (123).
4.2.6 Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from mouse eye was isolated and purified using mirVana miRNA Isolation Kit (Life
Technologies, Carlsbad, CA) according to the protocol of the manufacturer. Quantitative real-
time RT-PCR using primers synthesized at Integrated DNA Technologies were used for gene
expression analysis as described previously (123-125). RT-PCR was conducted using the
following primers: GAPDH (forward (F), 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3';
reverse (R), 5'-CATGTAGGCC ATGAGGTCCACCAC-3'); VEGF (F, 5'-
GCGGGCTGCCTCGCAGT C-3'; R, 5'-TCACCGCCTTGGCTTGTCAC- 3'). Amplification of
genes was performed with Bio-Rad real-time PCR system using One-Step SYBR® Green qRT-
PCR Kit and a standard thermal cycler protocol. The threshold cycle (CT) was determined, and
relative quantification was calculated by the comparative CT method as described previously
(124).
4.2.7 Statistical analysis
One-way ANOVA analysis followed by Tukey post-hoc test was performed. Additionally, when
appropriate, a two-way ANOVA analysis followed by Bonferroni posttest was performed.
GraphPad Prism (GraphPad Software Inc., San Diego, CA) was used for analyzing and plotting
the data (n=3) and p<0.05 was used as a significance criteria.
48
4.3 Results
4.3.1 CNV induction
Figure 4.1(A) shows images of the retinal/choroidal vasculature of a control mouse and a CNV-
induced mouse at 2 weeks post CNV induction. Normal blood vessels were seen in the control
mouse (left panel), whereas neovascularization and leaky blood vessels were evident in the
CNV-induced mouse (right panel). Figure 4.1(B) shows a comparison between the area of
retinal/choroidal blood vessels in the control eye vs. the CNV-induced eye. In the control eye,
vasculature comprised of 14.9% of the surface area; this value increased to 21.8% in the CNV-
induced sample indicating the significant increase in retinal/choroidal blood vessels retaining
fluorescein.
Figure 4.1: (A) FITC-dextran labeled retinal/choroidal flat-mount. Control (left) and CNV-induced eye (right). Magnification of 20x. (B) Total area of blood vessels in control and CNV-induced eye. Data is represented by mean ± SD (n=3). *p<0.05.
A B
49
4.3.2 Effect of corticosteroid drug delivery systems on CNV area
Figure 4.2 shows a comparison of neovascularization in retina/choroid tissue measured for
various treatment groups after 2 weeks of exposure. The untreated CNV-induced group showed a
mean percentage of 20.6%. Exposure to loteprednol etabonate (LE) and LE drug delivery system
(DDS) resulted in a significant decrease to 8.5% and 7.4% in the residual CNV area,
respectively. There was no significant difference between LE and LE DDS. Treatment with
intravitreal administration of triamcinolone acetonide (TA) solution had no effect on the CNV
area; however, TA DDS displayed a significant decrease in the CNV area.
Figure 4.2: Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area at 2 weeks. Groups tested include CNV-induced (no treatment), loteprednol etabonate (LE), LE drug delivery system (DDS), triamcinolone acetonide (TA), TA DDS. Data is represented by mean ± SD (n=3). *p<0.05 vs CNV.
50
The effect of the corticosteroids and DDS 4 weeks post treatment is presented in Figure 4.3.
Neither LE nor TA showed any significant reduction in CNV area. Exposure to LE DDS resulted
in a significant decrease in CNV area to 15.7% and exposure to TA DDS to 12.0%. This was
most likely due to the sustained drug release properties of the DDS.
Figure 4.3: Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area at 4 weeks. Groups tested include CNV-induced (no treatment), loteprednol etabonate (LE), LE drug delivery system (DDS), triamcinolone acetonide (TA), TA DDS. Data is represented by mean ± SD (n=3). *p<0.05 vs. CNV.
51
To study the sustained efficacy of the DDS, the CNV area of LE and LE DDS were compared
between 2 and 4 weeks (Figure 4.4). The CNV area of the LE treated group increased from 8.5%
to 22.8% and the LE DDS treated groups increased from 7.3% to 15.7% over the same period of
time.
Figure 4.4: Comparison of sustained efficacy of LE and LE DDS on CNV area at 2 and 4 weeks. Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area. Data is represented by mean ± SD (n=3). *p<0.05.
52
In Figure 4.5, the CNV area of TA-treated group increased from 16.6% at 2 weeks post-CNV
induction to 22.3% at 4 weeks. The TA DDS group increased from 7.2% to 13.6%; however, this
increase was not significant (p>0.05).
Figure 4.5: Comparison of sustained efficacy of TA and TA DDS on CNV area at 2 and 4 weeks. Effect of corticosteroids and corticosteroid drug delivery systems on retinal/choroidal neovascularization area. Data is represented by mean ± SD (n=3). *p<0.05.
53
4.3.3 Effect of corticosteroid drug delivery systems on VEGF expression
Gene expression of VEGF was analyzed in whole eye homogenates at 4 weeks post-treatment.
Figure 4.6 shows that TA DDS significantly reduced VEGF expression as compared to the CNV-
induced group.
Figure 4.6: Effect of corticosteroids and corticosteroid drug delivery systems on VEGF expression at 4 weeks. Data is represented by mean ± SD (n=3). *p<0.05 vs. CNV.
4.4 Discussion
CNV is a pathophysiological characteristic of neovascular AMD. Thus, neovascular
AMD is a disease which is characterized by increased levels of angiogenic factors in the
retina/choroid. These factors could be designated as targets in developing effective drug delivery
systems to mitigate CNV.
54
In this study, we evaluated the effect of corticosteroid drug delivery systems (DDS) on
the VEGF levels and the extent of neovascularization estimated by an image analysis in a mouse
model of CNV. Neovascularization was stimulated using a subretinal injection of PEG-8 to
activate the complement system (122), which in turn induces the development of new blood
vessels in the retina/choroid. A process of complement activation leading to CNV is the deposit
of membrane attack complex on the RPE and choroid; this initiates production and secretion of
VEGF as well as induces inflammation (122, 126, 127). Figure 4-1 depicted the FITC-dextran
labeled blood vessels in a control and untreated CNV-induced eye. The control eye demonstrated
regular network of blood vessels, whereas in the CNV-induced group, there was evidence of
neovascularization and leaky blood vessels. Quantification of the blood vessels showed a
significant increase in area from 14.9% in the control to 21.8% in the CNV-induced group.
We studied two corticosteroids with varying potencies: triamcinolone acetonide (TA) and
loteprednol etabonate (LE). TA is categorized as an upper-mid strength potency steroid; it is
commonly used in combination with other treatments for posterior ocular disorders (26). TA has
been indicated experimentally as an alternative option for the treatment of AMD alone or
combined with anti-VEGF agents (128). One additional advantage of TA is its prolonged
duration of action due to its low water solubility (128). However, adverse effects such as an
increase in intraocular pressure and cataract development can occur with higher potency steroids.
Therefore, we also examined LE as it was designed to reduce these side effects by converting
into an inactive metabolite and minimizing toxicity (107, 108). LE was reported to have a
therapeutic index 20-fold better than high potency corticosteroids such as hydrocortisone or
betamethasone in vivo (105). We tried to determine if a DDS formulation of LE would be
beneficial in maintaining a balance between the active duration and lower side effects. Figures
4.2 and 4.3 showed the effect on CNV area 2 and 4 weeks post treatment, respectively. 2 weeks
after treatment of CNV injury, LE and LE DDS resulted in a similar reduction of CNV area. This
is most likely due to the high relative potency of LE. TA drug solution alone showed no
significant change, whereas TA DDS elicited a significant reduction of CNV area, most likely
due to the sustained release of the DDS. At 4 weeks after treatment, the LE and TA drug solution
treatment groups no longer appeared to be suppressing the excess vasculature growth; however
the LE DDS and TA DDS still showed a significant reduction.
55
Figures 4.4 and 4.5 compare the results between the drug solution and the DDS at two
time points. While LE DDS showed a reduction in the occurrence of CNV, the comparison test
between week 2 and 4 showed that there was a significant increase in vascularization by week 4.
The inability of LE DDS to inhibit progression of the disease state might be due to the
conversion of LE to an inactive carboxylic acid metabolite after in vivo interaction. Once
released, it has been reported that the half-life of LE is 2.8 hours when administered systemically
(129). Further testing to optimize the formulation for enhanced release rate might improve the
long-term delivery. A comparison between TA and TA DDS showed no significant difference
between week 2 and 4, suggesting that TA maintained its therapeutic effect in the eye over the
course of one month when formulated in nanoparticles within a thermoreversible gel. These data
corroborate previously reported studies in TA NPs were more effective at reducing CNV than LE
NPs in a mouse CNV model (24).
Vascular endothelial growth factor (VEGF) is a well-known mediator of angiogenesis
(120, 122). Figure 4.6 showed the results from gene expression analysis for VEGF levels in the
eye after 4-week exposure to 4 different treatment groups. VEGF mRNA expression levels were
consistent with reported literature after CNV induction in experimental models (126). Only
treatment with TA DDS resulted in a significant decrease in VEGF levels. Surprisingly, LE DDS
did not have an effect on VEGF expression at 4 weeks; this is most likely why the CNV area had
significantly increased in the LE DDS treated groups between weeks 2 and 4.
4.5 Conclusions
Our data supports existing evidence that some corticosteroids released by DDS in the eye
can inhibit the progression of neovascularization in a CNV mouse model more effectively
compared to drug solutions alone.
Two corticosteroids were studied: LE, as a representative of the newer generation highly
potent corticosteroids and TA, as a representative of the older generation upper mid potent
corticosteroids. Our study demonstrated a significant reduction in CNV area at 2 weeks
compared to the CNV-induced eye by LE, while TA failed to inhibit neovascularization in the
same model. At 4 weeks, neither of the corticosteroids alone was effective in reducing the CNV
area compared to CNV-induced eye without any treatment.
56
Both LE DDS and TA DDS exhibited significant reduction of CNV area in 2 weeks
compared to CNV–induced eye. At 2 weeks, TA DDS showed significant reduction in CNV area
compared to drug alone while LE DDS system showed reduction in CNV area similar to drug
alone. At 4 weeks, both LE DDS and TA DDS were significantly effective in reducing CNV area
compared to drug alone while LE and TA alone were ineffective. This was likely due to
sustained drug release properties of the DDS.
Gene expression analysis of TA DDS indicated a significant reduction of VEGF
expression compared to the CNV-induced model as well as the other treatment groups. This
implies that TA could suppress angiogenesis via VEGF, and the DDS allows for sustained
release to maintain the effects of VEGF suppression for at least 4 weeks.
TA DDS was found to be a more effective delivery system at reducing CNV area after 2
weeks and 4 weeks and consequently, reducing the need for frequent intravitreal injections of the
corticosteroids.
The proposed DDS composed of PLGA-PEG nanoparticles in PLGA thermoreversible
gels can be useful in delivering other anti-VEGF antibodies agents such as bevacizumamb
(Avastin®), Ranizumamb (Lucentis®), and Aflibercept (Elyea®) for sustained delivery in the
posterior segment of the eye and reducing the frequency of painful intravitreal injection and
warrants further studies.
57
Chapter 5. Conclusions and Future Work
5.1. Summary and Conclusions
Macular degeneration affects 11 million people in the United States. Current anti-angiogenic
treatments require frequent intravitreal injections to maintain therapeutic intraocular doses to
suppress disease progression. Due to the lengthy pipeline for creation of new
pharmaceuticals, it makes sense to reposition approved drugs in novel drug delivery systems
to improve their sustained efficacy. In this study, we hypothesized that sustained-release
corticosteroid-nanoparticles incorporated in thermoreversible gels can improve the
therapeutic nature of corticosteroids for CNV in wet AMD.
In Chapter 2, “Triamcinolone Acetonide Nanoparticles Incorporated in Thermoreversible
Gels for Age-Related Macular Degeneration,” we prepared a DDS consisting of TA
nanoparticles and a thermoreversible gel for use in mitigating VEGF overexpression in vitro.
We characterized NPs for size and polydispersity index. The NPs were around 200 nm in
size with a polydispersity index less than 1, indicative of a unimodal size distribution. The
20% w/v thermoreversible gel displayed sol-gel transition at a temperature range appropriate
for physiological use. In vitro release assay demonstrated a sustained release; while the TA
drug solution alone was completely released within 48 hours, 31.49% of the drug was
released from the DDS in 10 days. The combined DDS displayed no cytotoxicity on human
ARPE-19 retinal pigment epithelial cells and was able to reduce the expression of VEGF in
vitro over 72 hours.
In Chapter 3, “Efficacy of Loteprednol Etabonate Drug Delivery System in Suppression of in
vitro Retinal Pigment Epithelium Activation,” we utilized a similar drug delivery system to
deliver LE, a soft steroid that elicits lower side effects in vivo. The LE DDS formulation
exhibited a sustained release pattern with 5% of the drug released over 10 days. Compared to
LE alone, LE DDS significantly reduced the expression of VEGF in ARPE-19 cells over 72
hours. The biocompatibility, low toxicity and therapeutic potential of the DDS make it an
ideal model for further investigation in a mouse CNV model.
58
Finally, in Chapter 4, “The Effect of Corticosteroid-Nanoparticles Incorporated in a
Thermoreversible Gel on Chemically Induced Choroidal Neovascularization in Mice,” the
corticosteroid DDS were tested on a CNV mouse model. Our data reinforces the in vitro
data that LE and TA in sustained DDS can inhibit the progression of neovascularization. LE
DDS and TA DDS effectively reduced the CNV area over 4 weeks compared to their
respective drug solution alone. These results lend themselves to further studies of clinical
relevance to utilize DDS composed of PLGA-PEG nanoparticles in PLGA thermoreversible
gels for posterior segment ocular disease.
5.2. Future Directions
The overall goal of this work is to produce a sustained release drug delivery system to
reduce the frequency of intravitreal injections necessary to control choroidal
neovascularization evident in neovascular age-related macular degeneration. The reported
study has shown promising results and warrants further studies to improve efficacy of the
DDS. Further extensions of the work presented would involve 1) investigation of factors
that would alter drug release from the nanoparticles, such as drug loading, particle size, ratio
of polymers; 2) analysis of molecular mechanisms affected by DDS; and 3) utilization of
combination therapy with anti-VEGF agents to enhance therapeutic effect of DDS.
5.2.1. Effect of factors that would alter drug release from DDS
Different parameters can affect the size of NPs, such as drug/polymer ratio, stabilizer and
homogenization (47) and a change in polymer mixture or ratio can affect the drug release
characteristics (94). In preliminary studies, we optimized the drug/polymer ratio to obtain
NPs that would be an appropriate size range for localization within the RPE. Additionally,
the NPs dispersed in the thermoreversible gel show a sustained release for up to one month.
Other studies have shown that polymer blending including PLGA of different molecular
weights can allow for higher drug entrapment due to closer packing of the polymers (94).
This parameters can be altered to incorporate more drug and further lengthen the sustained
release effects of the DDS.
59
5.2.2. Effect of DDS on molecular mechanisms of CNV
The in vivo study was designed to investigate the sustained efficacy of the DDS on the
incidence of CNV and effect on VEGF expression. However, it does not include analysis of
other angiogenic and inflammatory mediators that are involved in the progression of CNV.
CNV is associated with increased production of VEGF, TGF-β, and β-FGF (125).
Therefore, it would be important to investigate if exposure to the DDS results in a reduction
of these angiogenic factors by measuring mRNA and protein expression. Additionally,
analysis of inflammatory mediators such as MMPs, IL-6, TNF-α that are indicated in CNV
as well as inhibitors of angiogenesis such as angiostatin, endostatin, pigment epithelium-
derived factor (PEDF) would give valuable insight into the mechanisms by which our DDS
affects a response (14).
5.2.3. Combination therapy with corticosteroid DDS and anti-vascular endothelial growth
factor (VEGF) for an enhanced anti-angiogenic effect
Clinical therapy for neovascular macular degeneration is targeted against the vascular
component of choroidal neovascularization. Primary treatment includes anti-VEGF agents
that can halt the progression of angiogenesis as well as reduce vascular permeability. The
anti-angiogenic drugs currently used in the treatment of neovascular AMD work by binding
VEGF-A isoforms: ranibizumab (Lucentis), bevacizumab (Avastin), pegaptanib sodium
(Macugen) (7, 19-21). Corticosteroids are sometimes used to supplement these drugs to
enhance the anti-inflammatory effects (14). The DDS can include similar combination
therapy by utilizing both one of the anti-VEGF agents and either TA or LE. This system will
be more specific towards angiogenesis and have an enhanced anti-angiogenic effect as
compared to monotherapy (130). Effect on choroidal neovascularization area, drug
concentration in ocular tissue and effect on gene and protein expression would be measured.
60
References 1. Prevalence of Adult Vision Impairment and Age-Related Eye Diseases in America Bethesda, MD: National Eye Institue; 2014 [cited 2014 04/01/14]. Available from: http://www.nei.nih.gov/eyedata/adultvision_usa.asp. 2. Regatieri CV, Dreyfuss JL, Nader HB. Experimental Treatments for Neovascular Age-Related Macular Degeneration Ying G, editor. Shanghai, China: InTech; 2012. p. 83-98. 3. Thrimawithana TR, Young S, Bunt CR, Green C, Alany RG. Drug delivery to the posterior segment of the eye. Drug Discovery Today. 2011 Mar;16(5-6):270-7. PubMed PMID: 21167306. 4. Nirmal HB, Bakliwal SR, Pawar SP. In-situ gel: new trends in controlled and sustained drug delivery system. International Journal of PharmTech Research. 2010;2(2):1398-408. 5. Choi BG, Park MH, Cho SH, Joo MK, Oh HJ, Kim EH, et al. In situ thermal gelling polypeptide for chondrocytes 3D culture. Biomaterials. 2010 Dec;31(35):9266-72. PubMed PMID: 20864172. 6. Tamboli V, Mishra G, Mitra A. Biodegradable polymers for ocular drug delivery. In: Mitra A, editor. Advances in Ocular Drug Delivery2012. p. 65-86. 7. Bylsma GW, Guymer RH. Treatment of age-related macular degeneration. Clinical & Experimental Optometry: Journal of the Australian Optometrical Association. 2005 Sep;88(5):322-34. PubMed PMID: 16255691. 8. Normal Macula Clarksburg, MD: BrightFocus Foundation; 2000 [cited 2014 April 4, 2014]. Available from: http://www.brightfocus.org/macular/about/understanding/normal-macula.html. 9. Rofagha S, Bhisitkul RB, Boyer DS, Sadda SR, Zhang K, Group S-US. Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP). Ophthalmology. 2013 Nov;120(11):2292-9. PubMed PMID: 23642856. 10. Heier JS, Brown DM, Chong V, Korobelnik JF, Kaiser PK, Nguyen QD, et al. Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration. Ophthalmology. 2012 Dec;119(12):2537-48. PubMed PMID: 23084240. 11. Park YG, Rhu HW, Kang S, Roh YJ. New approach of anti-VEGF agents for age-related macular degeneration. Journal of Ophthalmology. 2012;2012:637316. PubMed PMID: 22496964. Pubmed Central PMCID: 3307057. 12. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nature Medicine. 2003 Jun;9(6):669-76. PubMed PMID: 12778165. 13. Verteporfin In Photodynamic Therapy Study G. Verteporfin therapy of subfoveal choroidal neovascularization in age-related macular degeneration: two-year results of a randomized clinical trial including lesions with occult with no classic choroidal neovascularization--verteporfin in photodynamic therapy report 2. American Journal of Ophthalmology. 2001 May;131(5):541-60. PubMed PMID: 11336929. 14. Campa C, Costagliola C, Incorvaia C, Sheridan C, Semeraro F, De Nadai K, et al. Inflammatory mediators and angiogenic factors in choroidal neovascularization: pathogenetic interactions and therapeutic implications. Mediators of Inflammation. 2010;2010. PubMed PMID: 20871825. Pubmed Central PMCID: 2943126.
61
15. Group MPS. Laser photocoagulation of subfoveal neovascular lesions of age-related macular degeneration: updated findings from two clinical trials. Archives of ophthalmology. 1993;111(9):1200. 16. Ishikawa M, Jin D, Sawada Y, Abe S, Yoshitomi T. Future therapies of wet age-related macular degeneration. Journal of Ophthalmology. 2015;2015:138070. PubMed PMID: 25802751. Pubmed Central PMCID: 4354726. 17. Smith AG, Kaiser PK. Emerging treatments for wet age-related macular degeneration. Expert Opinion on Emerging Drugs. 2014 Mar;19(1):157-64. PubMed PMID: 24555421. 18. Kaiser PK. Emerging therapies for neovascular age-related macular degeneration: drugs in the pipeline. Ophthalmology. 2013 May;120(5 Suppl):S11-5. PubMed PMID: 23642781. 19. Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, et al. Ranibizumab for neovascular age-related macular degeneration. The New England Journal of Medicine. 2006 Oct 5;355(14):1419-31. PubMed PMID: 17021318. 20. Melnikova I. Wet age-related macular degeneration. Nature Reviews Drug discovery. 2005 Sep;4(9):711-2. PubMed PMID: 16178119. 21. Vadlapudi AD, Ashaben P, Kishore C, Ashim KM. Recent patents on emerging therapeutics for the treatment of glaucoma, age-related macular degeneration and uveitis. Recent Patents on Biomedical Engineering. 2012;5(1):83-101. 22. Chen G, Li W, Tzekov R, Jiang F, Mao S, Tong Y. Bevacizumab versus ranibizumab for neovascular age-related macular degeneration: a meta-analysis of randomized controlled trials. Retina. 2015 Feb;35(2):187-93. PubMed PMID: 25105318. 23. Chen G, Li W, Tzekov R, Jiang F, Mao S, Tong Y. Ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema: a meta-analysis of randomized controlled trials. PloS ONE. 2014;9(12):e115797. PubMed PMID: 25541937. Pubmed Central PMCID: 4277392. 24. Mudunuri K. Intravitreal delivery of corticosteroid nanoparticles [Ph.D.]. Gainesville, FL: University of Florida; 2008. 25. Samudre SS, Lattanzio FA, Jr., Williams PB, Sheppard JD, Jr. Comparison of topical steroids for acute anterior uveitis. Journal of Ocular Pharmacology and Therapeutics. 2004 Dec;20(6):533-47. PubMed PMID: 15684812. 26. Wang Y, Wang VM, Chan CC. The role of anti-inflammatory agents in age-related macular degeneration (AMD) treatment. Eye. 2011 Feb;25(2):127-39. PubMed PMID: 21183941. Pubmed Central PMCID: 3044916. 27. Ciulla TA, Walker JD, Fong DS, Criswell MH. Corticosteroids in posterior segment disease: an update on new delivery systems and new indications. Current Opinion in Ophthalmology. 2004 Jun;15(3):211-20. PubMed PMID: 15118508. 28. Sherif Z, Pleyer U. Corticosteroids in ophthalmology: past-present-future. Ophthalmologica Journal International D'ophtalmologie. 2002 Sep-Oct;216(5):305-15. PubMed PMID: 12424394. 29. Wu WS, Wang FS, Yang KD, Huang CC, Kuo YR. Dexamethasone induction of keloid regression through effective suppression of VEGF expression and keloid fibroblast proliferation. The Journal of Investigative Dermatology. 2006 Jun;126(6):1264-71. PubMed PMID: 16575391.
62
30. Penfold PL, Wen L, Madigan MC, Gillies MC, King NJ, Provis JM. Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration. Clinical and Experimental Immunology. 2000 Sep;121(3):458-65. PubMed PMID: 10971511. Pubmed Central PMCID: 1905725. 31. Wang YS, Friedrichs U, Eichler W, Hoffmann S, Wiedemann P. Inhibitory effects of triamcinolone acetonide on bFGF-induced migration and tube formation in choroidal microvascular endothelial cells. Graefe's Archive for Clinical and Experimental Ophthalmology. 2002 Jan;240(1):42-8. PubMed PMID: 11954780. 32. Sendrowski DP, Jaanus SD, Semes LP, Stern ME. Anti-inflammatory drugs. 5th ed. Bartlett JD, Jaanus SD, editors. New York, NY: Elsevier Health Sciences; 2008. 816 p. 33. McGhee CN, Dean S, Danesh-Meyer H. Locally administered ocular corticosteroids: benefits and risks. Drug safety: an International Journal of Medical Toxicology and Drug Experience. 2002;25(1):33-55. PubMed PMID: 11820911. 34. Tzekov R, Lin T, Zhang KM, Jackson B, Oyejide A, Orilla W, et al. Ocular changes after photodynamic therapy. Investigative Ophthalmology & Visual Science. 2006 Jan;47(1):377-85. PubMed PMID: 16384988. 35. Kaiser PK, Boyer DS, Cruess AF, Slakter JS, Pilz S, Weisberger A, et al. Verteporfin plus ranibizumab for choroidal neovascularization in age-related macular degeneration: twelve-month results of the DENALI study. Ophthalmology. 2012 May;119(5):1001-10. PubMed PMID: 22444829. 36. Gambhire S, Bhalerao K, Singh S. In situ hydrogel: different approaches to ocular drug delivery. International Journal of Pharmacy and Pharmaceutical Sciences. 2013;5(2):27-36. 37. Zhang W, Prausnitz MR, Edwards A. Model of transient drug diffusion across cornea. Journal of Controlled Release. 2004 Sep 30;99(2):241-58. PubMed PMID: 15380634. 38. Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Advanced Drug Delivery Reviews. 2006 Nov 15;58(11):1131-5. PubMed PMID: 17097758. 39. Kleinberg TT, Tzekov RT, Stein L, Ravi N, Kaushal S. Vitreous substitutes: a comprehensive review. Survey of Ophthalmology. 2011 Jul-Aug;56(4):300-23. PubMed PMID: 21601902. 40. Peyman GA, Lad EM, Moshfeghi DM. Intravitreal injection of therapeutic agents. Retina. 2009 Jul-Aug;29(7):875-912. PubMed PMID: 19584648. 41. Pahuja P, Arora S, Pawar P. Ocular drug delivery system: a reference to natural polymers. Expert Opinion on Drug Delivery. 2012 Jul;9(7):837-61. PubMed PMID: 22703523. 42. Yasukawa T, Ogura Y, Tabata Y, Kimura H, Wiedemann P, Honda Y. Drug delivery systems for vitreoretinal diseases. Progress in Retinal and Eye Research. 2004 May;23(3):253-81. PubMed PMID: 15177203. 43. Saraiya NV, Goldstein DA. Dexamethasone for ocular inflammation. Expert Opinion on Pharmacotherapy. 2011 May;12(7):1127-31. PubMed PMID: 21457057. 44. Gaudana R, Ananthula HK, Parenky A, Mitra AK. Ocular drug delivery. The AAPS Journal. 2010 Sep;12(3):348-60. PubMed PMID: 20437123. Pubmed Central PMCID: 2895432.
63
45. Zhou HY, Hao JL, Wang S, Zheng Y, Zhang WS. Nanoparticles in the ocular drug delivery. International Journal of Ophthalmology. 2013;6(3):390-6. PubMed PMID: 23826539. Pubmed Central PMCID: 3693026. 46. Patel A, Cholkar K, Agrahari V, Mitra AK. Ocular drug delivery systems: An overview. World Journal of Pharmacology. 2013;2(2):47-64. 47. Sabzevari A, Adibkia K, Hashemi H, Hedayatfar A, Mohsenzadeh N, Atyabi F, et al. Polymeric triamcinolone acetonide nanoparticles as a new alternative in the treatment of uveitis: in vitro and in vivo studies. European Journal of Pharmaceutics and Biopharmaceutics. eV. 2013 May;84(1):63-71. PubMed PMID: 23295645. 48. Bu HZ, Gukasyan HJ, Goulet L, Lou XJ, Xiang C, Koudriakova T. Ocular disposition, pharmacokinetics, efficacy and safety of nanoparticle-formulated ophthalmic drugs. Current Drug Metabolism. 2007 Feb;8(2):91-107. PubMed PMID: 17305490. 49. Bourges JL, Gautier SE, Delie F, Bejjani RA, Jeanny JC, Gurny R, et al. Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles. Investigative Ophthalmology and Visual Science. 2003 Aug;44(8):3562-9. PubMed PMID: 12882808. 50. Bala I, Hariharan S, Kumar MN. PLGA nanoparticles in drug delivery: the state of the art. Critical Reviews in Therapeutic Drug Carrier Systems. 2004;21(5):387-422. PubMed PMID: 15719481. 51. Geldenhuys W, Mbimba T, Bui T, Harrison K, Sutariya V. Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers. Journal of Drug Targeting. 2011 Nov;19(9):837-45. PubMed PMID: 21692650. 52. Carroll RT, Bhatia D, Geldenhuys W, Bhatia R, Miladore N, Bishayee A, et al. Brain-targeted delivery of Tempol-loaded nanoparticles for neurological disorders. Journal of Drug Targeting. 2010 Nov;18(9):665-74. PubMed PMID: 20158436. 53. Kreppel F, Kochanek S. Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide. Molecular Therapy. 2008 Jan;16(1):16-29. PubMed PMID: 17912234. 54. Li F, Hurley B, Liu Y, Leonard B, Griffith M. Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. The Open Ophthalmology Journal. 2012;6:54-8. PubMed PMID: 22798970. Pubmed Central PMCID: 3394187. 55. Park D, Shah V, Rauck BM, Friberg TR, Wang Y. An anti-angiogenic reverse thermal gel as a drug-delivery system for age-related wet macular degeneration. Macromolecular Bioscience. 2013 Apr;13(4):464-9. PubMed PMID: 23316011. 56. Rauck BM, Friberg TR, Mendez CAM, Park D, Shah V, Bilonick RA, et al. Biocompatible reverse thermal gel sustains the release of intravitreal bevacizumab in vivo. Investigative Ophthalmology and Visual Science. 2014 Jan;55(1):469-76. PubMed PMID: WOS:000331877200055. English. 57. Vodithala S, Khatry S, Shastri N, Sadanandam DM. Development and evaluation of thermoreversible ocular gels of ketorolac tromethamine. International Journal of Biopharmaceutics. 2010;1(1):39-45. 58. Sutariya V, Miladore N, Geldenhuys W, Bhatia D, Wehrung D, Nakamura H. Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery. Pharmaceutical Development and Technology. 2013 Jul-Aug;18(4):957-62. PubMed PMID: 22206499.
64
59. Gopal L, Sharma T. Use of intravitreal injection of triamcinolone acetonide in the treatment of age-related macular degeneration. Indian Journal of Ophthalmology. 2007;55(6):431. 60. Sherif Z, Pleyer U. Corticosteroids in ophthalmology: past–present–future. Ophthalmologica Journal International D'ophtalmologie. 2002;216(5):305-15. 61. Wu WS, Wang FS, Yang KD, Huang C-C, Kuo Y-R. Dexamethasone induction of keloid regression through effective suppression of VEGF expression and keloid fibroblast proliferation. Journal of Investigative Dermatology. 2006;126(6):1264-71. 62. Wang YS, Friedrichs U, Eichler W, Hoffmann S, Wiedemann P. Inhibitory effects of triamcinolone acetonide on bFGF-induced migration and tube formation in choroidal microvascular endothelial cells. Graefe's Archive for Clinical and Experimental Ophthalmology. 2002;240(1):42-8. 63. Ciulla TA, Criswell MH, Danis RP, Hill TE. Intravitreal triamcinolone acetonide inhibits choroidal neovascularization in a laser-treated rat model. Archives of Ophthalmology. 2001;119(3):399-404. 64. Spaide RF, Sorenson J, Maranan L. Combined photodynamic therapy with verteporfin and intravitreal triamcinolone acetonide for choroidal neovascularization. Ophthalmology. 2003;110(8):1517-25. 65. McGhee CN, Dean S, Danesh-Meyer H. Locally administered ocular corticosteroids. Drug Safety. 2002;25(1):33-55. 66. Fernández-Robredo P, Sancho A, Johnen S, Recalde S, Gama N, Thumann G, et al. Current treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering. Journal of Ophthalmology. 2014;2014:1-13. 67. Nowak JZ. Age-related macular degeneration (AMD): pathogenesis and therapy. Pharmacological Reports. 2006;58(3):353-63. 68. Regatieri C, Dreyfuss J, Nader H. Experimental treatments for neovascular age-related macular degeneration. In: Ying G-S, editor. Age Related Macular Degeneration - The Recent Advances in Basic Research and Clinical Care. Rijeka, Croatia: InTech 2012. p. 83-98. 69. Buschini E, Piras A, Nuzzi R, Vercelli A. Age related macular degeneration and drusen: neuroinflammation in the retina. Progress in Neurobiology. 2011;95(1):14-25. 70. Bylsma GW, Guymer RH. Treatment of age‐related macular degeneration. Clinical and Experimental Optometry. 2005;88(5):322-34. 71. R Thrimawithana T, Young S, R Bunt C, R Green C, G Alany R. Drug delivery to the posterior segment of the eye: challenges and opportunities. Drug Delivery Letters. 2011;1(1):40-4. 72. Park YG, Rhu HW, Kang S, Roh YJ. New Approach of Anti-VEGF Agents for Age-Related Macular Degeneration. Journal of ophthalmology. 2012;2012. 73. Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nature Medicine. 2003;9(6):669-76. 74. Zhang W, Prausnitz MR, Edwards A. Model of transient drug diffusion across cornea. Journal of Controlled Release. 2004;99(2):241-58. 75. Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Advanced Drug Delivery Reviews. 2006;58(11):1131-5.
65
76. Gambhire S, Bhalerao K, Singh S. In situ hydrogel: different approaches to ocular drug delivery. International Journal of Pharmacy & Pharmaceutical Sciences. 2013;5(2):27-36. 77. Gaudana R, Ananthula HK, Parenky A, Mitra AK. Ocular drug delivery. The AAPS Journal. 2010;12(3):348-60. 78. Zhou HY, Hao JL, Wang S, Zheng Y, Zhang WS. Nanoparticles in the ocular drug delivery. International Journal of Ophthalmology. 2013;6(3):390. 79. Patel A, Cholkar K, Agrahari V, Mitra AK. Ocular drug delivery systems: an overview. World. 2013;2(2):47-64. 80. Sabzevari A, Adibkia K, Hashemi H, Hedayatfar A, Mohsenzadeh N, Atyabi F, et al. Polymeric triamcinolone acetonide nanoparticles as a new alternative in the treatment of uveitis: In vitro and in vivo studies. European Journal of Pharmaceutics and Biopharmaceutics. 2013;84(1):63-71. 81. Bu H-Z, Gukasyan HJ, Goulet L, Lou X-J, Xiang C, Koudriakova T. Ocular disposition, pharmacokinetics, efficacy and safety of nanoparticle-formulated ophthalmic drugs. Current Drug Metabolism. 2007;8(2):91-107. 82. Bourges J-L, Gautier SE, Delie F, Bejjani RA, Jeanny J-C, Gurny R, et al. Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles. Investigative Ophthalmology and Visual Science. 2003;44(8):3562-9. 83. Bala I, Hariharan S, Kumar MR. PLGA nanoparticles in drug delivery: the state of the art. Critical Reviews™ in Therapeutic Drug Carrier Systems. 2004;21(5):387-422. 84. Geldenhuys W, Mbimba T, Bui T, Harrison K, Sutariya V. Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers. Journal of Drug Targeting. 2011;19(9):837-45. 85. Carroll RT, Bhatia D, Geldenhuys W, Bhatia R, Miladore N, Bishayee A, et al. Brain-targeted delivery of Tempol-loaded nanoparticles for neurological disorders. Journal of Drug Targeting. 2010;18(9):665-74. 86. Kreppel F, Kochanek S. Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide. Molecular Therapy. 2008;16(1):16-29. 87. Li F, Hurley B, Liu Y, Leonard B, Griffith M. Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. The Open Ophthalmology Journal. 2012;6:54. 88. Nirmal H, Bakliwal S, Pawar S. In-Situ gel: New trends in controlled and sustained drug delivery system. International Journal of PharmTech Research. 2010;2(2):1398-408. 89. Choi BG, Park MH, Cho S-H, Joo MK, Oh HJ, Kim EH, et al. In situ thermal gelling polypeptide for chondrocytes 3D culture. Biomaterials. 2010;31(35):9266-72. 90. Park D, Shah V, Rauck BM, Friberg TR, Wang Y. An anti‐angiogenic reverse thermal gel as a drug‐delivery system for age‐related wet macular degeneration. Macromolecular Bioscience. 2013;13(4):464-9. 91. Vodithala S, Khatry S, Shastri N, Sadanandam M. Development and evaluation of thermoreversible ocular gels of ketorolac tromethamine. International Journal of Biopharmaceutics. 2010;1(1):39-45. 92. Sutariya V, Miladore N, Geldenhuys W, Bhatia D, Wehrung D, Nakamura H. Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for
66
glaucoma filtration surgery. Pharmaceutical Development and Technology. 2013;18(4):957-62. 93. Majithiya RJ, Ghosh PK, Umrethia ML, Murthy RS. Thermoreversible-mucoadhesive gel for nasal delivery of sumatriptan. AAPS PharmSciTech. 2006;7(3):E80-E6. 94. Duvvuri S, Janoria KG, Mitra AK. Development of a novel formulation containing poly (d, l-lactide-co-glycolide) microspheres dispersed in PLGA–PEG–PLGA gel for sustained delivery of ganciclovir. Journal of Controlled Release. 2005;108(2):282-93. 95. Moghimipour E, Tafaghodi M, Balouchi A, Handali S. Formulation and in vitro evaluation of topical liposomal gel of triamcinolone acetonide. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2013;4(1):101-7. 96. Gómez-Gaete C, Tsapis N, Besnard M, Bochot A, Fattal E. Encapsulation of dexamethasone into biodegradable polymeric nanoparticles. International Journal of Pharmaceutics. 2007;331(2):153-9. 97. Budhian A, Siegel SJ, Winey KI. Haloperidol-loaded PLGA nanoparticles: systematic study of particle size and drug content. International Journal of Pharmaceutics. 2007;336(2):367-75. 98. Patel SP, Vaishya R, Mishra GP, Tamboli V, Pal D, Mitra AK. Tailor-made pentablock copolymer based formulation for sustained ocular delivery of protein therapeutics. Journal of Drug Delivery. 2014;2014:401747. PubMed PMID: 25045540. Pubmed Central PMCID: 4090486. 99. Tamboli V, Mishra GP, Mitra AK. Novel pentablock copolymer (PLA-PCL-PEG-PCL-PLA) based nanoparticles for controlled drug delivery: Effect of copolymer compositions on the crystallinity of copolymers and in vitro drug release profile from nanoparticles. Colloid and Polymer Science. 2013 May 1;291(5):1235-45. PubMed PMID: 23626400. Pubmed Central PMCID: 3633208. 100. Suen WL, Chau Y. Specific uptake of folate-decorated triamcinolone-encapsulating nanoparticles by retinal pigment epithelium cells enhances and prolongs antiangiogenic activity. Journal of Controlled Release. 2013 Apr 10;167(1):21-8. PubMed PMID: 23313961. 101. Yoncheva K, Lambov N. Development of biodegradable poly (alpha-methylmalate) microspheres. Die Pharmazie. 2000;55(2):148-50. 102. Yasukawa T, Ogura Y, Tabata Y, Kimura H, Wiedemann P, Honda Y. Drug delivery systems for vitreoretinal diseases. Progress in Retinal and Eye Research. 2004;23(3):253-81. 103. Kadam RS, Tyagi P, Edelhauser HF, Kompella UB. Influence of choroidal neovascularization and biodegradable polymeric particle size on transscleral sustained delivery of triamcinolone acetonide. International Journal of Pharmaceutics. 2012 Sep 15;434(1-2):140-7. PubMed PMID: 22633904. Pubmed Central PMCID: 3573139. 104. Coffey MJ, DeCory HH, Lane SS. Development of a non-settling gel formulation of 0.5% loteprednol etabonate for anti-inflammatory use as an ophthalmic drop. Clinical Ophthalmology. 2013;7:299-312. 105. Comstock TL, Decory HH. Advances in corticosteroid therapy for ocular inflammation: loteprednol etabonate. International Journal of Inflammation. 2012;2012:789623. PubMed PMID: 22536546. Pubmed Central PMCID: 3321285. 106. Comstock TL, Paterno MR, Singh A, Erb T, Davis E. Safety and efficacy of loteprednol etabonate ophthalmic ointment 0.5% for the treatment of inflammation and pain following
67
cataract surgery. Clinical Ophthalmology. 2011;5:177-86. PubMed PMID: 21383946. Pubmed Central PMCID: 3045067. 107. Bodor N, Bodor N, Wu WM. A comparison of intraocular pressure elevating activity of loteprednol etabonate and dexamethasone in rabbits. Current Eye Research. 1992 Jun;11(6):525-30. PubMed PMID: 1505197. 108. Bodor N, Loftsson T, Wu WM. Metabolism, distribution, and transdermal permeation of a soft corticosteroid, loteprednol etabonate. Pharmaceutical Research. 1992 Oct;9(10):1275-8. PubMed PMID: 1448425. 109. Ilyas H, Slonim CB, Braswell GR, Favetta JR, Schulman M. Long-term safety of loteprednol etabonate 0.2% in the treatment of seasonal and perennial allergic conjunctivitis. Eye and Contact Lens. 2004 Jan;30(1):10-3. PubMed PMID: 14722462. 110. Controlled evaluation of loteprednol etabonate and prednisolone acetate in the treatment of acute anterior uveitis. American Journal of Ophthalmology. 1999 5//;127(5):537-44. 111. Bourges JL, Gautier SE, Delie F, Bejjani RA, Jeanny JC, Gurny R, et al. Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles. Investigative Ophthalmology and Visual Science. 2003;44:3562-9. 112. Bejjani RA, BenEzra D, Cohen H, Rieger J, Andrieu C, Jeaanny JC, et al. Nanoparticles for gene delivery to retinal pigment epithelial cells. Molecular Vision. 2005;11:124-32. 113. Normand N, Valamanesh F, Savoldelli M, Mascarelli F, BenEzra D, Courtois Y, et al. VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo. Molecular Vision. 2005;11:184-91. 114. Kassem MA, AbdelRahman AA, Ghorab MM, Ahmed MB, Khalil RM. Nanosuspensions as an ophthalmic delivery system for certain glucocorticoid drugs. Internation Journal of Pharmaceutics. 2007;340:126-33. 115. Silva GRd, Fialho SL, Siqueira RC, Jorge R, Junior AdSC. Implants as drug delivery devices for the treatment of eye diseases. Brazillian Journal of Pharmaceutical Sciences. 2010;46(3):585-95. 116. Hirani A, Grover A, Lee YW, Pathak Y, Sutariya V. Triamcinolone acetonide nanoparticles incorporated in thermoreversible gels for age-related macular degeneration. Pharmaceutical Development and Technology. 2014 Sep 26:1-7. PubMed PMID: 25259682. 117. Coleman HR, Chan CC, Ferris FL, 3rd, Chew EY. Age-related macular degeneration. Lancet. 2008 Nov 22;372(9652):1835-45. PubMed PMID: 19027484. Pubmed Central PMCID: 2603424. 118. Campochiaro PA. Retinal and choroidal neovascularization. Journal of Cellular Physiology. 2000 Sep;184(3):301-10. PubMed PMID: 10911360. 119. Necela BM, Cidlowski JA. Mechanisms of glucocorticoid receptor action in noninflammatory and inflammatory cells. Proceedings of the American Thoracic Society. 2004;1(3):239-46. PubMed PMID: 16113441. 120. Zhang X, Bao S, Lai D, Rapkins RW, Gillies MC. Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas. Diabetes. 2008 Apr;57(4):1026-33. PubMed PMID: 18174522. Pubmed Central PMCID: 2836241. 121. Zhang X, Wang N, Schachat AP, Bao S, Gillies MC. Glucocorticoids: structure, signaling and molecular mechanisms in the treatment of diabetic retinopathy and diabetic
68
macular edema. Current Molecular Medicine. 2014 Mar;14(3):376-84. PubMed PMID: 24467200. 122. Lyzogubov VV, Tytarenko RG, Liu J, Bora NS, Bora PS. Polyethylene glycol (PEG)-induced mouse model of choroidal neovascularization. The Journal of Biological Chemistry. 2011 May 6;286(18):16229-37. PubMed PMID: 21454496. Pubmed Central PMCID: 3091230. 123. Lambert V, Lecomte J, Hansen S, Blacher S, Gonzalez ML, Struman I, et al. Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nature Protocols. 2013 Nov;8(11):2197-211. PubMed PMID: 24136346. Epub 2013/10/19. 124. Lee WH, Sonntag WE, Mitschelen M, Yan H, Lee YW. Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain. International Journal of Radiation Biology. 2010 Feb;86(2):132-44. PubMed PMID: 20148699. Pubmed Central PMCID: 2827151. 125. Bora PS, Sohn JH, Cruz JM, Jha P, Nishihori H, Wang Y, et al. Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization. Journal of Immunology. 2005 Jan 1;174(1):491-7. PubMed PMID: 15611275. 126. Lyzogubov VV, Tytarenko RG, Jha P, Liu J, Bora NS, Bora PS. Role of ocular complement factor H in a murine model of choroidal neovascularization. The American Journal of Pathology. 2010 Oct;177(4):1870-80. PubMed PMID: 20813971. Pubmed Central PMCID: 2947282. 127. Anderson DH, Radeke MJ, Gallo NB, Chapin EA, Johnson PT, Curletti CR, et al. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited. Progress in Retinal and Eye Research. 2010 Mar;29(2):95-112. PubMed PMID: 19961953. Pubmed Central PMCID: 3641842. 128. Sarao V, Veritti D, Boscia F, Lanzetta P. Intravitreal steroids for the treatment of retinal diseases. The Scientific World Journal. 2014;2014:989501. PubMed PMID: 24526927. Pubmed Central PMCID: 3910383. 129. Hochhaus G, Chen LS, Ratka A, Druzgala P, Howes J, Bodor N, et al. Pharmacokinetic characterization and tissue distribution of the new glucocorticoid soft drug loteprednol etabonate in rats and dogs. Journal of Pharmaceutical Sciences. 1992 Dec;81(12):1210-5. PubMed PMID: 1491342. 130. Stewart MW. PDGF: Ophthalmology's Next Great Target. Expert Reviews in Ophthalmology. 2013;8(6):527-37.
69
Appendix A: Ocular Toxicity of Nanoparticles Anjali Hirani1,2* and Aditya Grover1, Yong W. Lee2, Vijaykumar Sutariya1, Yashwant Pathak1
1 Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida,
Tampa, FL 33612
2 School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA 24060
*A. Grover and A. Hirani are equal contributors to this work
Corresponding Author: Yashwant Pathak
Published: Hirani A, Grover A, Lee YW, Sutariya V, Pathak Y. (2014). Ocular Toxicity of
Nanoparticles In V. Sutariya and Y. Pathak (Eds.), Biointeraction of Nanomaterials (pp. 347-
352) Boca Raton, FL: CRC Press Taylor & Francis Group.
70
Contents
1.1. Use of Nanoparticles in Ocular Therapy
1.1.1. Nanoceria
1.1.2. CK30PEG
1.1.3. Magnetic Nanoparticles (MNP)
1.1.4. Chitosan
1.1.5. PLGA
1.1.6. Others
1.1.6.1. PACA
1.1.6.2. PECL
1.1.6.3. Nanomicelles
1.1.6.4. PCEP
1.1.6.5. Acrylate copolymers
1.1.6.6. Solid lipid nanoparticles (SLN)
1.2. References
71
1.1 Use of Nanoparticles in Ocular Therapy
The application of biodegradable nanoparticles in ocular therapies is of utmost
importance to the field of ocular medicine. The enhancement of current methods of therapy
could mean a drastic change in the quality of life of people suffering from a number of
degenerative posterior eye disorders. Currently, the most common form of ocular therapy
involved topical treatment in the form of eye drops due to its ease of use, minimal risk of
infection, and patient compliability (1). This method is limited in its effect, as natural processes
in the eye flush the drug out of the tissue within the first minute of application; lacrimation is one
such example (1, 2). The structural barriers in ocular tissue combined with the difficulty in drug
delivery make the posterior eye chamber a potentially neglected site of therapy (1).
To directly target the posterior eye chamber, a common method of therapy is intravitreal
injection (IVT) to deliver drugs to the retina (1, 2). This method is not without any adverse side
effects, common ones of which include tissue damage and infections (1). Nonbiodegradable
forms of treatment are also used, by which a nano-sized device is surgically implanted at the site
of therapy (3). The drawbacks of such a method are the relative large sized incision required to
implant a device and the repeated implantations of a new device once the previous one has
exhausted its drug supply; neglecting removal of the device may cause it to be encapsulated by
fibrous tissue (3). Possible complications with this type of therapy include retinal detachment,
vitreous hemorrhage, and dissolution of the device, among others (3).
It behooves pharmaceutical researchers to develop nanotechnologies that bypass these
invasive methods. Such particles improve tissue penetration, bypassing IVT, and provide
sustained drug or gene therapy, a possible advantage that would bypass the need for viral gene
therapy, a method that is not without its own risk of infection and hemorrhagic complications (2,
4, 5). By being able to control the dramatic increase in surface area to volume ratio of
nanoparticles as compared to comparable macroscopic devices, researchers would be able to
enhance tissue penetration and drug delivery systems directly to the effected tissue (4, 6).
The accessibility of the eye makes it a great target for nanoparticle therapy (2). However,
such technologies are not without their own drawbacks. Nanoparticles may be coated with toxic
72
chemicals which could be released into the body during therapies or may build up into tissues
and cause blockages leading to an increase in interocular pressure (3, 4). The retina and optic
neural tissue are very sensitive to toxic materials, which may cause unforeseen complications
due to seepage of toxic materials during therapy (2, 5, 6).
The most common forms of nanoparticle toxicity that are expressed in ocular tissue are
oxidative stress, counteractions with cell membranes, and inflammation (2). These parameters
were tested in the trials conducted with the following nanoparticles outlined in this section of the
chapter. Because the rabbit is the most common animal model used for ocular toxicity studies,
most of the studies cited used this animal to obtain toxicity data (2). The large lens of the mouse
and rat make them poor models, as administrating intraocular injections proves difficult (2). In
addition, a number of ocular complications arise in the mouse or rat by even the slightest touch
of the eye by the needle, including inflammation or the development of cataracts (2). Monkeys’
eyes provide the best model for studying ocular toxicity of nanoparticles, but none of the
following studies cited used the monkey as a model for the respective toxicity studies; it is
unsure why not (2).
1.1.1 Nanoceria
One of the causes of retinal degenerative diseases, such as diabetic retinopathy, is
oxidative damage due to reactive oxygen radical species (6). Cerium oxide nanoparticles, also
known as nanoceria, have been developed as antioxidants and free radical scavengers as a
potential therapy for such neurodegenerative diseases (6). When these nanoparticles are
synthesized in the 3-5 nm range, they mimic the effects of the antioxidant enzymes, such as
superoxide dismutase and catalase, which neutralize superoxide anions and hydrogen peroxides,
respectively (6-8).
The primary method of administration for the nanoceria particles was via IVT. After
injection, the retinal tissue accumulated the greatest concentration of nanoparticles and 70% of
the particles were retained 120 days post-injection (6). Nanoceria was not actively eliminated
from the eye, as 90% of the injected nanoceria was retained in the eye 120 days post-injection
(6). The experimental half-life yield of the nanoceria in the retina was 414 days with a half-life
73
of 525 days in the eye. Studies by the Asati et al. showed that the polymer coating of the
nanoceria particles may induce a charge on the particles, varying the rates of uptake in different
tissues and inducing localization in certain tissues (6, 9). They also found that positively charged
nanoceria particles could be taken up by more cell types than negatively charged particles (9).
Previous studies showed that weekly injected nanoceria did not have cytotoxic effects in
heart, kidney, brain, lung, spleen, and liver; cytotoxic studies in ocular tissue also showed that
nanoceria did not have toxic effects on healthy retinal tissue over a range of doses (6).
Investigations of four types of retinal tissue – superior and inferior central retina and superior
and inferior peripheral retina – 9 days post-IVT injection showed that there was no reduction in
the thickness of these layers. This finding along with the lack of observable change in retinal
function post nanoceria IVT injection as compared to saline injected eyes further suggests that
nanoceria does not have any short- or long-term cytotoxic effects on retinal tissue (6). In
addition, nanoceria particles were shown to be nontoxic to optical neural tissue up to 120 days
post-injection (6).
However, there may be some drawbacks to using nanoceria particles. Synthesis of
nanoceria particles through use of hexamethylenetetramine (HMT) may induce cytotoxic effects
in ocular tissue (6). In addition, some cell culture studies with nanoceria have yielded results of
particle aggregation, which may negatively affect ocular tissue (10). Given these possible
negative effects of nanoceria particles, there are no negative effects on healthy retinal cells with
enhanced redox nanoceria capacity (6). The minimal cytotoxic effects of nanoceria particles on
retinal tissue make it a great candidate for possible widespread ocular drug therapies.
1.1.2 CK30PEG
Recombinant adeno-associated viruses (AAV) have been extensively used in ocular
tissue as a gene therapeutic method by which long-term gene expression can be induced, thereby
offering therapeutic intervention for defects in large genes (11, 12). Plasmid DNA compacted
with polyethylene glycol (PEG)-substituted polylysine (CK30PEG) nanoparticles offer a
promising alternative to AAV gene therapies (13, 14). Subretinal injections of CK30PEG
nanoparticles were able to induce persistent gene expression in mice for up to a year, opening up
74
doors to efficiently deliver up to 20 kbp plasmid vectors efficiently to dividing and post-mitotic
cells (11, 13, 15). These nanoparticles have been shown to be safe and effective in human
clinical trials, thereby allowing for the direct targeting of molecular markers in photoreceptors
and retinal pigment epithelium cells for gene therapies (11). Similar therapeutic results have been
shown in mouse models expressing the retinitis pigmentosa phenotype (16).
Histological examination showed that the subretinal delivery of CK30PEG nanoparticles
did not induce infiltration of inflammatory cells in the eye (13). Injected eyes did not show the
proliferation of polymorphonuclear leukocytes (PMN), an early response to toxicity (13).
Myeloperoxidase (MPO) immunoreactivity, an activator of inflammatory signaling cascades,
was not detected in injected eyes, nor was F4/80 microglia/macrophage marker, a response to
ischemia-induced retinopathy (13). PMN, MPO, and F4/80 markers were histologically
expressed in positive experimental controls (13). The lack of such expression in CK30PEG
injected eyes suggests that these nanoparticles do not cause an inflammatory cascade in injected
eyes (13).
Enzyme-linked immunosorbent assay (ELISA) and Real-time reverse-transcription PCR
(qRT-PCR) were used to detect the presence of interleukin-8 (IL-8), monocyte chemotactic
protein-1 (MCP-1), and tumor necrosis factor- (TNF-α) proteins and mRNA, respectively, in
CK30PEG injected eyes, saline injected eyes, and Bacillus cereus endophthalmitis eyes as a
positive control (13). IL-8 can be produced in response to inflammatory stimuli and is involved
in the initiation and amplification of acute inflammatory response processes (13). B. cereus eyes
expressed elevated levels of IL-8 protein and mRNA, with lack of elevation of either expressed
by nanoparticle and saline injected eyes (13). MCP-1 is a member of the chemokine family and
recruits monocytes to sites of injury and infection (13). Eyes injected with CK30PEG
nanoparticles showed transient elevations in MCP-1 mRNA and protein which returned to saline-
injected control levels after 1 day (13). B. cereus positive control eye samples expressed
markedly elevated levels of MCP-1 protein and mRNA (13). TNF-α is produced by macrophages
and monocytes as a part of the inflammation cascade pathway and apoptotic cell death (13).
There was no detectable increase in TNF-α levels following subretinal injection of CK30PEG,
compared to significantly increased TNF-α levels in endophthalimitis positive control eyes.
75
The lack in expression of inflammatory cascade proteins in eyes with subretinally-
injected CK30PEG suggests that this gene therapy model is nontoxic in ocular tissue, thereby
safely inducing sustained gene expression in necessary tissues (13).
1.1.3 Magnetic Nanoparticles (MNP)
DNA-tethered magnetic nanoparticles are FDA-approved MRI contrast agents able to
successfully deliver genes to targeted ocular tissues (2, 4, 5). These nanoparticles are nontoxic to
retinal tissue; apart from successfully transfecting ocular cells, eyes treated with MNP did not
show signs of inflammation nor did they induce white blood cell infiltration, both intravitreally
and subretinally (5). Intraocular pressure in MNP treated eyes remained the same as PBS treated
eyes, suggesting that the particles did not disturb any intraocular meshwork (4).
Histological analysis of MNP treated ocular tissue did not yield any signs of iron oxide
toxicity by the nanoparticles (4). MTT cytotoxicity assays showed the biocompatibility of MNP
coated with polyethylenoxide copolymers, suggesting that these nanoparticles are safe for
intraocular injection (4). In addition, the iron oxide was not shown to cause oxidative stress in
vivo (2).
The use of uncoated MNP could lead to aggregation and oxidation in vivo, thus natural,
biocompatible and biodegradable polymers are used to coat the nanoparticles (4). Adaptation of
the surface of the particle could enhance product delivery to tissues and could target specific
tissues by the polymer coat used on the MNP (4). Because iron oxide is a component of the
MNP, exposure to external magnetic fields could be harmful to body tissues, however this data
was not provided (4). In addition, iron could damage photoreceptors in the eye, thereby causing a
serious side effect (4, 17). However, the low iron load in the MNP and the protective polymer
coating prevented iron from leaking out into the tissue and showed no great amplitude difference
in electroretinography (ERG) waveforms as compared to tissues injected with PBS (4). Because
MNP causes no major cytotoxicity issues in vivo for up to 5 months post-injection, MNP is one
of the safest nanoparticle gene delivery mechanisms currently available (2, 4).
1.1.4 Chitosan
76
Chitosan, a biocompatible and nontoxic deacetylated form of chitin derived from
crustacean shells, is one of the least expensive and most widely used nanomaterial in ocular
therapies (2, 5). The positively-charged surface of the nanoparticles helps it interact well with the
negatively-charged corneal surface and has been successful in delivering drugs and genes to
ocular tissue (2, 5).
Given these benefits, chitosan is a biomaterial that has different effects in different ocular
tissues, making it compatible in some and incompatible in others (2, 5). Topically, chitosan
shows biocompatibility and efficient gene delivery with little to none tolerance issues nor any
tissue necrosis up to 24 hours post treatment (2). However, chitosan induces acute inflammatory
responses when injected intravitreally (2, 5). The severe inflammatory response caused a vitreous
haze and membranous opacities caused by infiltration of a large number of monocytes to
phagocytize the foreign polysachharide-based chitosan nanoparticles, suggesting that the
immunomodulatory hyalocytes in the vitreous humor are particularly sensitive to chitosan (2, 5).
There were signs of retinal degradation at the sites of most severe inflammation (5). The
dichotomy in biological interaction of chitosan with different tissues suggests that chitosan is a
promising nanoparticle for topical ocular therapy but a poor intraocular therapeutic nanoparticle.
1.1.5 Polylactic-C-glycolic Acid (PLGA)
PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA) (3). This
biodegradable, biocompatible copolymer is one of the most studied polymers and is a popular
choice for the treatment of choroidal neovascularization (2, 3). The ratio of PLA and PGA in the
synthesis of PLGA can be altered to change the therapeutic effects of the copolymer in biological
systems by changing the total surface area, rate of drug release, and rate of polymer degradation
(3). Because PGA is synthesized using toxic solvents, improper formulations may cause toxicity
in biological systems (3). Furthermore, PLGA can be synthesized by emulsification in acetone
and methylene chloride, also chemicals that may induce cytotoxicity (2). However, dose-
dependent studies of PLGA in biological systems did not yield any signs of cytotoxic effects on
ocular tissues (2). With no reports of cytotoxicity in the eye, PLGA remains one of the most
widely used, FDA-approved nanoparticles for experimental nanotherapies (2).
77
1.1.6 Other Nanoparticles
1.1.6.1 Poly(alkyl-cyanoacrylate) (PACA)
PACA is a colloidal suspension of nanoparticles shown to prolong the corneal penetration
of hydrophilic and lipophilic drugs in the eye (18). These compounds have a strong shelf life, as
they maintained their mean size and appeared unchanged when stored at room temperature for 6
months (18). The corneal penetration may be due to their colloidal nature, however its
therapeutic application may be hindered by the disruption caused to the corneal cell membrane
(18).
1.1.6.2 Poly-ε-caprolactone (PECL) nanoparticles and nanocapsules
PECL is a hydrophobic, biodegradable, and biocompatible polymer of ε-caprolactone (3).
PECL nanoparticles are slowly broken down in biological systems by the hydrolysis of ester
linkages but the nanoparticles can be mixed with more hydrophilic polymers to form copolymers
that can be broken down at faster rates (3, 19). Experimental studies in rabbit eyes showed that
the nanoparticles are well tolerated without any signs of inflammation in anterior and posterior
segments of the eye (20).
PECL nanocapsules enhance the penetration of lipophilic drugs in ocular tissues without
damaging the cell membranes, with penetration rates more favorable than PECL nanoparticles
(18). The sizes of nanocapsules did not change after 6 months at room temperature, suggesting
that the polymer coating imparts stability to the nanocapsules (18).
1.1.6.3 Nanomicelles
Nanomicelles, up to 100 nm in size, are a low-toxicity colloidal dispersion of molecules
with a hydrophobic core and hydrophilic shell (1, 21). These molecules provide an excellent
method by which to solubilize hydrophobic drugs with therapeutic concentrations and administer
them to hydrophilic tissues, thereby lowering drug degradation and enhancing permeability (1).
Because the hydrophilic sclera is an efficient therapeutic pathway to the posterior eye,
nanomicelles make it easier for hydrophobic drugs to efficiently diffuse to the posterior eye with
minimal degradation (1). In addition, the hydrophilicity of the nanomicelle shell may confer
78
resistance against systemic circulation washout via ocular blood and lymphatic vessels (1). This
relatively new nanotherapeutic molecule has shown little toxicity in biological systems, but more
toxicological studies are warranted before its widespread use (1).
1.1.6.4 Poly[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide]
(PCEP)
PCEP is a DNA-condensing agent with gene therapy potential in the eye (5). Similarly to
MNP, PCEP did not induce inflammation when injected intravitreally or subretinally (5). These
relatively harmless molecules did not attract white blood cells to the site of injection (5). Because
of its inert, biodegradability, and low toxicity, it serves as a potential ocular nanoparticle
transfection agent (5).
1.1.6.5 Acrylate polymers (Eudragit)
Eudragit is a nanoparticle that improves drug stability and maximizes drug dosages and effects
(2). The biological activity of eudragit can be directly manipulated by the choice of polymer used
to make the eudragit copolymer (2). There were no reports of ocular toxicity after 10 minutes of
application, with mild irritation in the first 10 minutes reported by 20-30% of subjects (2).
Because of its low- to nontoxicity, eudragit serves as an ocular topically therapeutic nanoparticle.
1.1.6.6 Solid lipid nanoparticles (SLN)
SLN, ranging in size up to 400 nm, are biodegradable and biocompatible, and have been
used as nanoparticles since the 1990s (2). The advantage of these nanoparticles is their advanced
drug load that they can carry (2). However, due to the nature of its synthesis, a potential source
of toxicity may be the presence of excipients on the nanoparticle (2). In addition, some
surfactants dissociate from the nanoparticle during sterilization due to the high temperatures,
another potential source of toxicity; there is no dissociation of surfactants at body temperature
(2).
79
1.2 References
1. Vadlapudi AD, Mitra AK. Nanomicelles: an emerging platform for drug delivery to the
eye. Therapeutic Delivery. 2013;4(1):1-3.
2. Prow TW. Toxicity of nanomaterials to the eye. Wiley Interdisciplinary Reviews:
Nanomedicine and Nanobiotechnology. 2010;2(4):317-33.
3. Christoforidis JB, Chang S, Jiang A, Wang J, Cebulla CM. Intravitreal devices for the
treatment of vitreous inflammation. Mediators of Inflammation. 2012;2012.
4. Raju HB, Hu Y, Vedula A, Dubovy SR, Goldberg JL. Evaluation of magnetic micro-and
nanoparticle toxicity to ocular tissues. PLoS One. 2011;6(5):e17452.
5. Prow TW, Bhutto I, Kim SY, Grebe R, Merges C, McLeod DS, et al. Ocular nanoparticle
toxicity and transfection of the retina and retinal pigment epithelium. Nanomedicine:
Nanotechnology, Biology and Medicine. 2008;4(4):340-9.
6. Wong LL, Hirst SM, Pye QN, Reilly CM, Seal S, McGinnis JF. Catalytic nanoceria are
preferentially retained in the rat retina and are not cytotoxic after intravitreal injection. PLoS
One. 2013;8(3):e58431.
7. Jessica E. Nanoceria exhibit redox state-dependent catalase mimetic activity. Chemical
Communications. 2010;46(16):2736-8.
8. Self WT, Seal S. Nanoparticles of cerium oxide having superoxide dismutase activity.
Google Patents; 2009.
9. Asati A, Santra S, Kaittanis C, Perez JM. Surface-charge-dependent cell localization and
cytotoxicity of cerium oxide nanoparticles. ACS Nano. 2010;4(9):5321-31.
10. Verma A, Stellacci F. Effect of surface properties on nanoparticle–cell interactions.
Small. 2010;6(1):12-21.
11. Han Z, Conley SM, Makkia R, Guo J, Cooper MJ, Naash MI. Comparative analysis of
DNA nanoparticles and AAVs for ocular gene delivery. PLoS One. 2012;7(12):e52189.
12. Amado D, Mingozzi F, Hui D, Bennicelli JL, Wei Z, Chen Y, et al. Safety and efficacy of
subretinal readministration of a viral vector in large animals to treat congenital blindness.
Science Translational Medicine. 2010;2(21):21ra16.
13. Ding X-Q, Quiambao AB, Fitzgerald JB, Cooper MJ, Conley SM, Naash MI. Ocular
delivery of compacted DNA-nanoparticles does not elicit toxicity in the mouse retina. PLoS One.
2009;4(10):e7410.
80
14. Liu G, Li D, Pasumarthy MK, Kowalczyk TH, Gedeon CR, Hyatt SL, et al.
Nanoparticles of compacted DNA transfect postmitotic cells. Journal of Biological Chemistry.
2003;278(35):32578-86.
15. Fink T, Klepcyk P, Oette S, Gedeon C, Hyatt S, Kowalczyk T, et al. Plasmid size up to
20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles.
Gene Therapy. 2006;13(13):1048-51.
16. Cai X, Conley SM, Nash Z, Fliesler SJ, Cooper MJ, Naash MI. Gene delivery to mitotic
and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype
in a mouse model of retinitis pigmentosa. The FASEB Journal. 2010;24(4):1178-91.
17. Declercq SS, Meredith P, Rosenthal AR. Experimental siderosis in the rabbit: correlation
between electroretinography and histopathology. Archives of Ophthalmology. 1977;95(6):1051-
8.
18. Calvo P, Vila‐Jato JL, Alonso MJ. Comparative in vitro evaluation of several colloidal
systems, nanoparticles, nanocapsules, and nanoemulsions, as ocular drug carriers. Journal of
Pharmaceutical Sciences. 1996;85(5):530-6.
19. Pitt C. Poly-e-caprolactone and its copolymers. In: Langer MCaRS, editor. Biodegradable
Polymers as Drug Delivery Systems. New York, NY: Marcel Dekker; 1990. p. 71.
20. Silva-Cunha A, Fialho, SL., Naud, MC., Behar-Cohen, F. Poly-e-caprolactone
intravitreous devices: an in vivo study. Investigative Ophthalmology Visual Science.
2009;50(5):2312-8.
21. Trivedi R, Kompella UB. Nanomicellar formulations for sustained drug delivery:
strategies and underlying principles. Nanomedicine. 2010;5(3):485-505.
81
Appendix B: Nanotechnology for Omics-based Ocular Drug
Delivery
Anjali Hirani1,2,*, Aditya Grover1,*, Yong Woo Lee2, Yashwant Pathak1, Vijaykumar Sutariya1
1 Department of Pharmaceutical Sciences, USF College of Pharmacy, University of South
Florida, Tampa, FL 33612.
2 School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University,
Blacksburg, VA 24061.
* These authors contributed equally to this work.
This chapter/paper appears in Handbook of Research on Diverse Application of Nanotechnology
in Biomedicine, Chemistry and Engineering edited by S. Soni, A. Salhotra, and M. Suar
Copyright 2014, IGI Global, www.igi-global.com. Posted by permission of the publisher
Published: Hirani A, Lee YW, Pathak YV, Sutariya V. (2014). Nanotechnology for Omics-based
Ocular Drug Delivery In S. Soni, A. Salhotra, and M. Suar (Eds.), Handbook of Research on
Diverse Application of Nanotechnology in Biomedicine, Chemistry and Engineering (pp. 152-
166) Hershey, PA: IGI Global.
82
ABSTRACT
Millions of people suffer from ocular diseases that impair vision and can lead to blindness.
Advances in genomics and proteomics have revealed a number of different molecular markers
specific for different ocular diseases, thereby optimizing the processes of drug development and
discovery. Nanotechnology can increase the throughput of data obtained in omics-based studies
and allows for more sensitive diagnostic techniques as well more efficient drug delivery systems.
Biocompatible and biodegradable nanomaterials developed through omics-based research are
able to target reported molecular markers for different ocular diseases and offer novel
alternatives to conventional drug therapy. In this chapter, we review the pathophysiology, current
genomic and proteomic information and current nanomaterial-based therapies of four ocular
diseases: glaucoma, uveal melanoma, age-related macular degeneration, and diabetic
retinopathy. Omics-based research can be used to elucidate specific genes and proteins and
develop novel nanomedicine formulations to prevent, halt, or cure ocular diseases at the
transcriptional or translational level.
83
OCULAR DISEASE
Approximately 140 million Americans over the age of 40 suffer from a variety of ocular diseases
that impair vision and may lead to blindness (NEI, 2012). The prevalence of ocular disorders will
continue to increase with the worldwide aging population. Although current treatments do exist,
there is a need for better diagnostics and more efficient therapies that can arrest the progression
and/or even reverse damage of ocular diseases. Currently, more information is needed to
understand the pathogenesis of ocular disease as well as determining new drug targets to enhance
ocular drug discovery or repositioning current drugs for more efficacious therapy.
Limitations in Treatment for Ocular Diseases
Currently, therapies exist to delay progression of some ocular diseases; however, a better
understanding of pathogenic processes is needed to find more effective treatments. Some of the
common ocular diseases presented later in this chapter possess a complex interplay of genetic
factors that are challenging to treat. Discovery of genes responsible for ocular disorders can aid
in the development of new therapeutic agents. Clinically, we are merely treating symptoms and
not targeting the actual disease mechanisms. More research needs to be completed to elucidate
these mechanisms.
A number of barriers for ocular drug delivery exist, such as nasolacrimal drainage and the blood-
aqueous and blood-retinal barriers. This restricts administration of potential therapeutics. Drug
can be delivered by a variety of routes including topical ocular, periocular injection, intravitreal
injection, and systemic administration. The topical route is a convenient method of drug delivery
to the anterior segment; however, a model of transient diffusion has shown that less than 5% of a
lipophilic drug and 0.5% of a hydrophilic drug penetrate the cornea (W. Zhang, Prausnitz, &
Edwards, 2004), and the remainder is cleared through nasolacrimal drainage and systemic
absorption(Gambhire, Bhalerao, & Singh, 2013) . The amount of available drug that permeates
across the sclera is reduced with cationic and lipophilic solutes and the RPE has tight
intercellular junctions for hydrophilic molecules (Urtti, 2006). Additionally, the lymphatic
system, blood vessels and active transporters all work to clear drugs administered through
transscleral routes. Systemic administration of drugs requires high doses that are potentially toxic
to obtain a therapeutic concentration across the blood ocular barriers (Geroski & Edelhauser,
84
2000; Sigurdsson, Konraethsdottir, Loftsson, & Stefansson, 2007). Intravitreal injections
circumvent physiological barriers and maintain therapeutic doses without damage to bystander
tissues; however, frequent injections can lead to complications like retinal detachment, increase
in ocular pressure, and hemorrhage (Peyman, Lad, & Moshfeghi, 2009). Given the presence of
these physiological barriers, the development of therapies utilizing nanotechnology that
efficiently deliver drugs and extend drug release to the eye would be beneficial to the
progression of ocular disease treatment. Due to the lengthy pipeline in gaining FDA approval,
newly repositioned drugs can be utilized to expand current disease therapy.
Omics-based nanotechnology
The goal of omics-based nanotechnology is to use nanoscale technology to enhance early
detection, gain an understanding of pathophysiology, as well as find better treatments for eye
disease. Nanogenomics refers to a new approach for medical diagnostics and therapy (Nicolini,
2006). Nanoproteomics allows us to evaluate expression of ocular proteins, identify novel
therapeutic targets study the pharmacological effects of therapeutics (Steely & Clark, 2000).
These new techniques can improve the current understanding of ocular diseases and aid in the
discovery of therapies targeting genes responsible for ocular disease.
APPLICATION TO OCULAR DISEASES
Glaucoma
Pathophysiology:
Glaucoma causes blindness in about 7 million people every year, only 10% of the people
affected by glaucoma worldwide (Quigley, 1996). A number of risk factors have been identified
for glaucoma, including hypertension, family history, and age, among others (Abbot F Clark &
Thomas Yorio, 2003). Glaucoma is caused by progressive damage to the trabecular meshwork,
optic nerve head, and retinal ganglion cells; however, the exact mechanism behind the nerve
damage is yet unknown (Abbot F Clark & Thomas Yorio, 2003; Frank S Ong et al., 2013).
Glaucoma can usually be diagnosed as open- or closed-angle and primary or secondary based on
the degree of ocular hypertension (Abbot F Clark & Thomas Yorio, 2003). Elevated intraocular
pressure (IOP) is characteristic of primary open-angle glaucoma (POAG), the most common type
of glaucoma, and is the most common target of treatment.
85
Current genomic and proteomic information:
A number of genetic markers that play a role in multiple different types of glaucomas
have been identified.
MYOC was one of the first genes identified for juvenile and adult-onset POAG (Stone et al.,
1997). Its associated protein, myocilin, has been associated with the trabecular meshwork and
may be associated with increased outflow resistance, leading to an increase in IOP pressure
(Fautsch, Bahler, Jewison, & Johnson, 2000; WuDunn, 2002; Z. Zhou & Vollrath, 1999).
Reiger syndrome is a genetic disorder which manifests itself in craniofacial and umbilical
abnormalities (Amendt, Semina, & Alward, 2000). The varied phenotypes associated with
Reiger syndrome are associated with different mutations in the PITX2 gene and the PITX2
transcription factor for which it codes. More than half of the individuals diagnosed with Reiger
syndrome develop glaucoma, making the mutant PITX2 gene a risk factor and molecular marker
for glaucoma (WuDunn, 2002).
Primary congenital glaucoma has been traced to mutations in CYP1B1 gene. CYP1B1
codes for the CYP1B1 protein from the cytochrome P450 family, but its mechanism in ocular
pathology is not yet fully understood (Stoilov, Jansson, Sarfarazi, & Schenkman, 2001). Studies
of various ethnic populations affected with congenital glaucoma revealed multiple mutations of
the CYP1B1 gene responsible for the disease in Western- and Middle Eastern as well as
Japanese populations (Michels-Rautenstrauss et al., 2001).
Six genes (GLC1A-GLC1F) have been identified through pedigree analysis of families
affected by hereditary POAG. PCOLCE2 has also been identified as a possible indicator of
glaucoma because of its association with the trabecular meshwork and the GLC1C locus, but no
mutations in PCOLCE2 were identified in patients with GLC1C-induced POAG (WuDunn,
2002). Population studies in Estonian POAG patients also showed a significantly higher
association between individuals positive for the glutathione S-transferase (GSTM1) protein with
POAG as compared to a control population. Smoking increased this risk (Juronen et al., 2000).
Reductions in the outflow of aqueous humor from the anterior chamber of the eye have
been implicated in glaucoma pathology due to its association to IOP. Imbalances in the
expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs),
which remodel the extracellular matrix (ECM) of the trabecular meshwork, may account for the
differences in aqueous humor outflow in patients with glaucoma (Wong et al., 2002). Disruption
86
of trabecular meshwork ECM was also revealed as a result of increased TGF-beta levels in
glaucoma patients (Zhao, Ramsey, Stephan, & Russell, 2004). Prostaglandins increase the
activity of MMPs in ciliary smooth muscle cells, which effects the outflow of aqueous humor,
and can lead to a decrease in the high IOP pressure characteristic of POAG (Weinreb,
Kashiwagi, Kashiwagi, Tsukahara, & Lindsey, 1997). In addition, cochlin protein has been
identified as an elevator of IOP pressure in glaucoma patients by disrupting the trabecular
meshwork (Bhattacharya et al., 2005).
Current therapies:
A number of current nanomedicine therapies have been investigated in the treatment of
glaucoma physiology. The benefits that nanomedicines offer are their biodegradability, sustained
release of drugs, and targeting of necessary tissues and molecular pathways.
Chu et al. investigated the activity of 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT)-
loaded calcium phosphate nanoparticles (CAP) in the reduction of IOP and aqueous flow rate in
glaucomatic rabbits. 7-OH-DPAT in CAP showed a hypotensive, therefore therapeutic, effect in
the glaucomatic model; however, raclopride, a dopamine D2/D3 receptor agonist, was shown to
reduce the effect of 7-OH-DPAT in CAP. The data suggests that dopamine D2/D3 receptors may
play a role in modulating IOP and that CAP may potentially be a therapeutic agent for glaucoma.
In addition, further investigations of the role of the dopamine D2/D3 receptor in glaucoma may
elucidate further directed therapies for the modulation of glaucomatic IOP (Chu, He, & Potter,
2002).
Wadhwa et al. revealed the IOP-reducing effects of hyaluronic acid (HA)-conjugated
chitosan (CS) nanoparticles (CS-HP-NPs) loaded with dorzolamide hydrochloride and timolol
maleate, 2 drugs used in glaucoma treatment. CS-NPs have been used as nanomedicine carriers
due to their biodegrability and drug availability, but conjugation with HA synergistically
increases the mucoadhesion of CS-NPs. In vivo studies in rabbits revealed a significant IOP
reduction in rabbits treated with CS-HA-NPs as opposed to the free drug solution, suggesting its
potential use against glaucoma (Wadhwa, Paliwal, Paliwal, & Vyas, 2010).
Jiang et al. reported the neuroprotective role of glial cell-line derived neurotrophic factor
(GDNF) loaded into poly DL-lactide-co-glycolide (PLGA) microspheres in elevated IOP-
induced rats. Intravitreal injections of the GDNF-PLGA microspheres were significantly better in
87
the neuroprotection of retinal ganglion cells (RGCs) in chronically elevated IOP through the
analysis of glial fibrillary acidic protein (GFAP) production compared to the blank PLGA
microspheres and GDNF alone (Jiang et al., 2007). It is reasonable to believe that the
nanomedicine administration of various other genes and RNA inhibitors of proteins may be able
to provide further neuroprotection to RGCs after injury or surgery (Zarbin, Montemagno, Leary,
& Ritch, 2013). Indeed, as a potential post-operative therapy to protect against scarring after
glaucoma surgery, Dos Santos et al. investigated the role of nanosized complexes of antisense
TGF-beta2 phosphorothiorate oligonucleotides (PS-ODN) with polyethylenimine (PEI)
encapsulated in PLGA microspheres. Subconjunctival injections in rabbits significantly
improved bleb survival and increased the intracellular penetration of PS-ODN, revealing the
microspheres’ potential therapeutic role (Gomes dos Santos et al., 2006).
The incorporation of drugs, genes, or oligonucleotides into biodegradable and
biocompatible nanomedicine vectors may provide novel therapeutic potentials in the prevention
and treatment of glaucoma. The size range of nanomedicines make these vectors compatible for
endocytosis into effected cells and also offer sustained release of the compounds associated with
the vectors. These compounds may be able to directly influence the genetic and molecular
pathways that influence the glaucoma phenotype.
Uveal Melanoma
Pathophysiology:
Uveal melanoma (UM) is one of the most common ocular cancers in adults, with 7 cases
per million each year and over 20 cases per million in those over the age of 70 (Ramasamy et al.,
2014; Singh & Topham, 2003). Over ninety percent of UM cases arise in the choroid and have
the worst prognosis, followed by the ciliary body and iris, with the best prognosis (Ramasamy et
al., 2014). Over half of UM cases result in metastasis, 40% of which result in death even after
primary tumor treatment (Bedikian, 2006). Close to 90% of UM metastatic cases result in
metastasis to the liver; the skin, bones, and lungs are other common sites of metastasis following
metastasis to the liver (Lorigan, Wallace, & Mavligit, 1991; Singh & Borden, 2005).
88
Current genomic and proteomic information:
Heat shock protein 27 (HSP-27) is a cytoplasmic protein involved in cell migration,
cytoskeletal structure, cell survival, and tumor progression (Kostenko & Moens, 2009). It plays
different roles in a number of different cancers; HSP-27 is over expressed in gastric, prostate,
and node-negative breast carcinoma and indicates a poor prognosis in each, but over-expression
of HSP-27 indicates good prognosis in non-small-cell lung- and ovarian carcinomas (Ramasamy
et al., 2014). Proteomic analysis of primary UM tissue revealed a down regulation of HSP-27 in
monosomy 3 tumors, with a significantly lower expression in monosomy 3 tumors as opposed to
disomy 3 tumors (Coupland et al., 2010). In addition, Jmor et al. showed that a significantly
reduced expression of HSP-27 in UM tissue correlated with a predicted survival of less than 8
years (Jmor, Kalirai, Taktak, Damato, & Coupland, 2012). Investigations in a human cuteanous
melanoma cell line revealed that the over expression of HSP-27 inhibited cell proliferation and
reduced cell invasiveness, which suggests that the under expression of HSP-27 might induce
greater cell migration in UM (Aldrian et al., 2002).
Two-dimensional difference gel electrophoresis (2D DIGE) followed up by
immunohistochemical studies in primary UM tumor samples also identified the over expression
of fatty acid-binding protein heart type (FABP3) and triosephosphate isomerase (TPI1). In
addition, siRNA knockdowns of these 2 proteins in a primary UM cell line revealed significantly
reduced levels of cell invasion and migration (Linge et al., 2012). FABPs are thought to play a
number of metabolic roles in cells, including growth, differentiation, and apoptosis (Lichtenfels
et al., 2009). Investigation of the over expression of FABP in small cell lung cancer implicate its
role in mitosis and cell growth (L. Zhang, Cilley, & Chinoy, 2000). FABP has also been linked
with poor prognosis and tumor aggressiveness in human gastric carcinoma (Hashimoto et al.,
2004). TPI1 plays a role in the cell’s glycolysis and gluconeogenesis pathways, high rates of
which are required for tumor cells (Albery & Knowles, 1976; Bui & Thompson, 2006). TPI1 has
been reported to be associated with the aggressiveness of breast cancer, and its under expression
was found to induce apoptosis in the HeLa cell line (Lee et al., 2010; Selicharova et al., 2008).
However, its over expression in lung cancer tissue, cell lines, and plasma, as well as in prostate
cancer implicates its role in the progression of disease and as biomarkers (Chen et al., 2002;
Kim, Koo, Kim, Sohn, & Park, 2008; Qian et al., 2010). The identification of these genetic
markers in UM and their varied roles in a number of different types of cancers suggests that their
89
over expression in primary UM tumors may contribute to the oncogeneity of the tissue. Further
investigations of the roles of FABP3 and TPI1 in UM may reveal novel pathways for therapeutic
intervention as well as screening techniques in preventing the progression of UM.
Proteomic analyses of primary tumors and cell lines have revealed the above-mentioned proteins
as potential biomarkers for UM; however, further such studies are required to elucidate more
proteins for the identification of UM. There is a lack of genomic and proteomic analysis of UM
tissues in available literature, but such studies would greatly increase the understanding of the
molecular mechanisms behind the induction and progression of the disease. The proteins and
biomarkers identified by future studies would provide researchers with genomic and molecular
pathways to target through nanotechnological intervention.
Current therapies:
The relatively low incidences of UM and the considerable lack of extensive genomic and
proteomic studies of UM tissues contribute to low number of nanomedicines to target UM.
However, the versatile nature of nanomedicines suggests its possible successes in treating UM.
Wang et al. investigated the role of dendrimer nanoparticle transfection therapy in the
human choroidal melanoma cell line (OCM-1) as a potential treatment (Yingchih Wang, Mo,
Wei, & Shi, 2013). The dendrimer nanoparticles were complexed with recombinant DNA
plasmids of tumor necrosis factor-a (TNFa) and herpes simplex virus thymidine kinase (HSV-
TK), both constructed with the promoter sequence of early growth response-1 (Egr1). Dendrimer
nanoparticles are biocompatible and are formed from nanoparticle polymers with sizes of less
than 100 nm, physiologically relevant when treating ocular diseases. The polymer is composed
of amino groups which are protonated at physiological pH levels, allowing electrostatic
interactions with oligonucleotides and their compaction and protection during transfection. The
transfection complex is able to be endocytosed by the cell, allowing the oligonucleotide complex
to be released into the cell and enter the nucleus for transcriptional regulation. Dendrimer-based
nanoparticle transfection complexes offer biocompatibility and the protection of associated
nucleic acids (Yingchih Wang et al., 2013).
Molecular studies in various tumors have identified the HSV-TK suicide gene as a
promising and successful cancer treatment by killing infected cells and surrounding uninfected
cells when administered in conjunction with other drugs or compounds (Rainov, 2000). Wang et
90
al. administered HSV-TK with TNF-a due to the natural anti-tumor properties exhibited by TNF-
a, including the ability to activate immune responses and cause hemorrhaging and necrosis in
tumoric tissues (Ha Thi et al., 2013; Kearney et al., 2013). HSV-TK and TNF-a were complexed
with Egr1 promoter due to Egr1’s revealed anti-tumor properties when associated with target
genes (Y. Zhou et al., 2010) and the hypothesis that irradiation with 125I may activate the Egr1
promoter complex and induce the transcription and translation of TNF-a and HSV-TK (Yingchih
Wang et al., 2013).
Wang et al. radiated the successfully-transfected tissues with 125I and found a
significantly elevated expression of TNF-a and HSV-TK through western blot and ELISA
analysis after radiation as compared to before, thought to be due to the activation of Egr1
transcription. Transmission electron microscopy (TEM) analysis of irradiated TNF-TK
transfected tissues revealed an increased number of cells in the necrotic state along with the
inhibition of cell growth and proliferation. The successful coupling of radiation with gene
therapy revealed that targeted gene therapy through the Egr1 promoter and radiation could
provide for a novel therapeutic route against UM (Yingchih Wang et al., 2013).
Future proteomic studies that reveal a number of other proteins involved in UM
pathological pathways may also become similar targets for in vitro and in vivo experiments such
as the one conducted by Wang et al. Coupling nanomedicine gene therapy with standard
radiation therapy reveals a synergistic therapeutic effect in treated tissues, and may lead to
clinical breakthroughs and better therapies.
Age-Related Macular Degeneration
Pathophysiology:
Age-related macular degeneration (AMD) is a disease that destroys sharp, central vision. It
affects the central region of the retina known as the macula, which is responsible for fine vision.
It is characterized by the presence of drusen and area of hyperpigmentation on the retinal
pigment epithelium (RPE). Advanced AMD is characterized by choroidal neovascularization
(CNV), the growth of abnormal blood vessels beneath the RPE or between the RPE and the
retina(Bylsma & Guymer, 2005). It is accompanied by fluid and blood rupturing Bruch’s
membrane into the subretinal space, leading to irregularities of the retina.
91
Current genomic and proteomic information:
Case studies have provided evidence for a variety of genetic markers involved in the occurrence
of AMD, namely in the formation of drusen and degeneration of macula. Docosahexaenoic acid
(DHA) is present in photoreceptor cell membranes in the retina. It is extremely sensitive to
oxidative damage and cleavage results in the production of carboxyethylpyrrole (CEP), an
oxidative protein (Gu et al., 2010). CEP has been found to be abundant in ocular tissue of
patients presenting with AMD. While a genetic marker of oxidative damage, CEPs promote the
growth of capillaries and can contribute to neovascularization (Lu et al., 2009).
Due to the neovascularization present in AMD, anti-angiogenic therapy is useful to slow the
progression of the disease. Vascular endothelial growth factor A (VEGF-A) is the most potent
promoter of angiogenesis and vascular permeability and its role in the pathogenesis of
neovascular AMD is well recognized (Ferrara, Gerber, & LeCouter, 2003; Park, Rhu, Kang, &
Roh, 2012; Verteporfin In Photodynamic Therapy Study, 2001). VEGF levels and vitreous levels
are elevated in human CNV in compared to healthy controls (Sendrowski, Jaanus, Semes, &
Stern, 2008). Due to the implication of VEGF in the progression of AMD, anti-angiogenic drugs
have been recently pursued to block the development and leakage of new, abnormal blood
vessels.
Current therapies:
Three anti-angiogenic drugs are currently being used in the treatment of AMD: ranibizumab
(Lucentis), bevacizumab (Avastin), and pegaptanib (Macugen)(Bylsma & Guymer, 2005).
Ranibizumab is a human recombinant antibody fragment that displays high binding affinity
towards all VEGF isoforms. Clinical trials have shown that ranibizumab helps maintain stable
vision without further progression of the disease; however, because of the high cost of the drug,
the use of the drug worldwide is limited (Rosenfeld et al., 2006). Bevacizumab has been used
more recently as an ‘off label’ therapy. It is a full-length human recombinant monoclonal
antibody, which binds all VEGF isoforms. It is FDA approved for colorectal, lung, and breast
cancer, but is used in clinical trials for AMD due to lower patient cost. Pegaptanib is a pegylated
aptamer that acts as an anti-VEGF agent. It binds the VEGF165 isoform and inhibits
angiogenesis (Vadlapudi, Ashaben, Kishore, & Ashim, 2012). For these anti-angiogenic drugs,
the biggest challenge is route of administration. Intravitreal injections allow for the most direct
92
approach; however, the chronic nature of the disease requires consistent injections resulting in
side-effects such as retinal detachment and cataract formation (Mudunuri, 2008). Recent studies
using nanoemulsions and polymeric micelles containing anti-VEGF bevacizumab result in
sustained delivery. Additional research has shown that pDNA encapsulated by micelles can
ameliorate choroidal neovascularization in AMD (F. S. Ong et al., 2013). These nanoemulsions
and polymeric micelles used for delivery offer more effective drug delivery by their ability to
maintain therapeutic concentrations of the active drug molecule over a longer duration than
previous methods.
Diabetic Retinopathy
Pathophysiology:
Diabetic retinopathy (DR) is a consequence of diabetes, usually developing within 5-10 years.
The disease is associated with progressive retinal ischemia (A. F. Clark & T. Yorio, 2003). It is
characterized by loss of pericytes in retinal capillaries and subsequent thickening of capillary
basement membranes and local ischemia. This induces the expression of VEGF, which can cause
vessel leakage and formation of abnormal blood vessels, similar to AMD. The majority of vision
loss from DR is due to macular edema. Other factors such as proliferation of fibrovascular
membranes can lead to retinal detachment (A. F. Clark & T. Yorio, 2003).
Current genomic and proteomic information:
DR has been studied by proteomic analysis of the vitreous humor. Mass spectroscopy
based studies have shown that elevated levels of extracellular carbonic anhydrase-I and kallikrein
are present in patients with DR. This suggests that retinal hemorrhage contribute to the DR
proteomic information. Studies displaying intravitreal injection of carbonic anhydrase-I in rats
lead to increased retinal vessel leakage and edema (Gao et al., 2007).
Current therapies:
Current therapies include photocoagulation therapy and pharmacological agents that block
VEGF signaling, such as ruboxistaurin mesylate which is a protein kinase C- inhibitor.
Additionally, corticosteroids are also being evaluated for their angiostatic, antipermeable and
antifibrotic properties (Samudre, Lattanzio, Williams, & Sheppard, 2004; Y. Wang, Wang, &
Chan, 2011). Triamcinolone acetonide and dexamethasone are used in combination with other
93
treatments for posterior ocular disorders. They act by binding steroid receptors in cells to induce
or repress targeted genes, thereby inhibiting inflammatory symptoms like edema and vascular
permeability (Sherif & Pleyer, 2002). Corticosteroids act on VEGF by inhibiting VEGF secretion
and inhibiting cytokine production (Wu, Wang, Yang, Huang, & Kuo, 2006). Corticosteroids can
also inhibit basic fibroblast growth factor and ICAM-1 expression and decrease VEGF levels
that are evident in neovascularization (Penfold et al., 2000; Y. S. Wang, Friedrichs, Eichler,
Hoffmann, & Wiedemann, 2002). As with AMD, nanoparticles have gained attention as a drug
delivery system to overcome the blood-retinal barriers. Recent studies have encapsulated current
therapies in nanoemulsions to sustain drug delivery. Alternatively AuNPs alone have been
shown to inhibit retinal neovascularization by suppressing VEGF receptor activation. It is
believed that AuNP bind to the heparin-binding proteins of certain VEGF isoforms (Jo, Lee, &
Kim, 2011).
CONCLUSIONS AND FUTURE PERSPECTIVES
Genomic and proteomic analyses consist of a number of different techniques to help identify the
molecular basis behind diseases. Because the eyes act as our window to the world around us,
measures taken to protect the eyes from damage would be of great benefit to all people.
We have described a number of different ocular diseases, ranging in severity from slight
discomfort caused by IOP in glaucomatic patients, to partial blindness in AMD patients, leading
to death in patients of UM. We have described a number of molecular markers, genomic and
proteomic, that act as indicators for their respective diseases. These genes and proteins are
involved in a multitude of pathways in ocular tissues and may play different roles in different
diseases, but elucidating their role in ocular diseases helps in developing therapies against those
diseases. However, the data obtained from genomic and proteomic analyses in ocular tissues
available in current literature is not exhaustive; further research needs to be conducted to identify
many more omics-related markers in all kinds of ocular tissues and diseases.
What has been reported so far in regards to genomic and proteomic biomarkers of ocular
diseases have been useful in the development of nanomedicine for those diseases. Nanomedicine
offers a number of advantages when compared to conventional drug therapy, including small
sizes, tissue-targeting, sustained release of the drug, low toxicity, biocompatibility, and
biodegradability. Nanomedicine is between 1-999 nm in size, but most are around 100-200 nm in
94
size, sizes that are tolerated in ocular tissues. The sizes and targeting of tissues through surface
conjugations of the nanomedicine vectors enable their endocytosis into the cell. Once in the cell,
nanomedicine exhibits a steady and sustained release of the drug compound over days, weeks,
and even months. Most nanomedicine vectors are made from organic compounds that are well
tolerated in ocular tissues, such as PLGA, and can be broken down into harmless compounds by
catabolismand eventually excreted. The ability to deliver novel drug, nucleic acid, protein, and
herbal compounds through complexing or encapsulating them within nanomedicine allows for
the direct targeting of the molecular pathways indicated by omics analysis specific for that
disease. Through directly targeting specific proteins, the disease can be prevented, arrested, or
even cured at the transcriptional or translational level. Further and continued omics-based
research will have a direct impact on the well-being and prognosis of degenerative ocular
diseases through the development of novel nanomedicine formulations.
95
REFERENCES
Albery, W John, & Knowles, Jeremy R. (1976). Free-energy profile for the reaction catalyzed by
triosephosphate isomerase. Biochemistry, 15(25), 5627-5631.
Aldrian, Silke, Trautinger, Franz, Fröhlich, Ilse, Berger, Walter, Micksche, Michael, & Kindas-
Mügge, Ingela. (2002). Overexpression of Hsp27 affects the metastatic phenotype of
human melanoma cells in vitro. Cell stress & chaperones, 7(2), 177.
Amendt, BA, Semina, EV, & Alward, WLM. (2000). Rieger syndrome: a clinical, molecular,
and biochemical analysis. Cellular and Molecular Life Sciences CMLS, 57(11), 1652-
1666.
Bedikian, Agop Y. (2006). Metastatic uveal melanoma therapy: current options. International
ophthalmology clinics, 46(1), 151-166.
Bhattacharya, Sanjoy K, Rockwood, Edward J, Smith, Scott D, Bonilha, Vera L, Crabb, John S,
Kuchtey, Rachel W, . . . Crabb, John W. (2005). Proteomics reveal Cochlin deposits
associated with glaucomatous trabecular meshwork. Journal of Biological Chemistry,
280(7), 6080-6084.
Bui, Thi, & Thompson, Craig B. (2006). Cancer's sweet tooth. Cancer cell, 9(6), 419-420.
Bylsma, G. W., & Guymer, R. H. (2005). Treatment of age-related macular degeneration. Clin
Exp Optom, 88(5), 322-334.
Chen, Guoan, Gharib, Tarek G, Huang, Chiang-Ching, Thomas, Dafydd G, Shedden, Kerby A,
Taylor, Jeremy MG, . . . Iannettoni, Mark D. (2002). Proteomic analysis of lung
adenocarcinoma identification of a highly expressed set of proteins in tumors. Clinical
Cancer Research, 8(7), 2298-2305.
Chu, Teh-Ching, He, Qing, & Potter, David E. (2002). Biodegradable calcium phosphate
nanoparticles as a new vehicle for delivery of a potential ocular hypotensive agent.
Journal of ocular pharmacology and therapeutics, 18(6), 507-514.
Clark, A. F., & Yorio, T. (2003). Ophthalmic drug discovery. Nat Rev Drug Discov, 2(6), 448-
459. doi: 10.1038/nrd1106
Clark, Abbot F, & Yorio, Thomas. (2003). Ophthalmic drug discovery. Nature Reviews Drug
Discovery, 2(6), 448-459.
96
Coupland, Sarah E, Vorum, Henrik, Mandal, Nakul, Kalirai, Helen, Honoré, Bent, Urbak, Steen
Fiil, . . . Damato, Bertil. (2010). Proteomics of uveal melanomas suggests HSP-27 as a
possible surrogate marker of chromosome 3 loss. Investigative ophthalmology & visual
science, 51(1), 12-20.
Fautsch, Michael P, Bahler, Cindy K, Jewison, David J, & Johnson, Douglas H. (2000).
Recombinant TIGR/MYOC increases outflow resistance in the human anterior segment.
Investigative ophthalmology & visual science, 41(13), 4163-4168.
Ferrara, N., Gerber, H. P., & LeCouter, J. (2003). The biology of VEGF and its receptors. Nat
Med, 9(6), 669-676. doi: 10.1038/nm0603-669
Gambhire, S., Bhalerao, K., & Singh, S. (2013). In situ hydrogel: Different approaches to ocular
drug delivery. Int. J. Pharm. Pharm Sci., 5(2), 27-36.
Gao, B. B., Clermont, A., Rook, S., Fonda, S. J., Srinivasan, V. J., Wojtkowski, M., . . . Feener,
E. P. (2007). Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral
vascular permeability through prekallikrein activation. Nat Med, 13(2), 181-188. doi:
10.1038/nm1534
Geroski, D. H., & Edelhauser, H. F. (2000). Drug delivery for posterior segment eye disease.
Invest Ophthalmol Vis Sci, 41(5), 961-964.
Gomes dos Santos, Ana L, Bochot, Amélie, Doyle, Aoife, Tsapis, Nicolas, Siepmann, Juergen,
Siepmann, Florence, . . . Fattal, Elias. (2006). Sustained release of nanosized complexes
of polyethylenimine and anti-TGF-β2 oligonucleotide improves the outcome of glaucoma
surgery. Journal of controlled release, 112(3), 369-381.
Gu, J., Pauer, G. J., Yue, X., Narendra, U., Sturgill, G. M., Bena, J., . . . Proteomic, A. M. D.
Study Group. (2010). Proteomic and genomic biomarkers for age-related macular
degeneration. Adv Exp Med Biol, 664, 411-417. doi: 10.1007/978-1-4419-1399-9_47
Ha Thi, Huyen Trang, Lim, Hee-Sun, Kim, Jooyoung, Kim, Young-Mi, Kim, Hye-Youn, &
Hong, Suntaek. (2013). Transcriptional and post-translational regulation of Bim is
essential for TGF-β and TNF-α-induced apoptosis of gastric cancer cell. Biochimica et
Biophysica Acta (BBA)-General Subjects, 1830(6), 3584-3592.
Hashimoto, Takeaki, Kusakabe, Takashi, Sugino, Takashi, Fukuda, Takeaki, Watanabe, Kazuo,
Sato, Yukio, . . . Fujii, Hiroshi. (2004). Expression of heart-type fatty acid-binding
97
protein in human gastric carcinoma and its association with tumor aggressiveness,
metastasis and poor prognosis. Pathobiology, 71(5), 267-273.
Jiang, Caihui, Moore, Michael J, Zhang, Xinmei, Klassen, Henry, Langer, Robert, & Young,
Michael. (2007). Intravitreal injections of GDNF-loaded biodegradable microspheres are
neuroprotective in a rat model of glaucoma. Mol Vis, 13, 1783-1792.
Jmor, Fidan, Kalirai, Helen, Taktak, Azzam, Damato, Bertil, & Coupland, Sarah E. (2012).
HSP‐27 protein expression in uveal melanoma: correlation with predicted survival.
Acta ophthalmologica, 90(6), 534-539.
Jo, D. H., Lee, T. G., & Kim, J. H. (2011). Nanotechnology and nanotoxicology in retinopathy.
Int J Mol Sci, 12(11), 8288-8301. doi: 10.3390/ijms12118288
Juronen, Erkki, Tasa, Gunnar, Veromann, Siiri, Parts, Lii, Tiidla, Anne, Pulges, Riina, . . .
Mikelsaar, Aavo-Valdur. (2000). Polymorphic glutathione S-transferase M1 is a risk
factor of primary open-angle glaucoma among Estonians. Experimental eye research,
71(5), 447-452.
Kearney, Conor J, Sheridan, Clare, Cullen, Sean P, Tynan, Graham A, Logue, Susan E, Afonina,
Inna S, . . . Martin, Seamus J. (2013). Inhibitor of apoptosis proteins (IAPs) and their
antagonists regulate spontaneous and tumor necrosis factor (TNF)-induced
proinflammatory cytokine and chemokine production. Journal of Biological Chemistry,
288(7), 4878-4890.
Kim, Jung Eun, Koo, Kyung Hee, Kim, Yeul Hong, Sohn, Jeongwon, & Park, Yun Gyu. (2008).
Identification of potential lung cancer biomarkers using an in vitro carcinogenesis model.
Experimental & molecular medicine, 40(6), 709-720.
Kostenko, Sergiy, & Moens, Ugo. (2009). Heat shock protein 27 phosphorylation: kinases,
phosphatases, functions and pathology. Cellular and molecular life sciences, 66(20),
3289-3307.
Lee, Won-Hee, Choi, Joon-Seok, Byun, Mi-Ran, Koo, Kyo-tan, Shin, Soona, Lee, Seung-Ki, &
Surh, Young-Joon. (2010). Functional inactivation of triosephosphate isomerase through
phosphorylation during etoposide-induced apoptosis in HeLa cells: Potential role of
Cdk2. Toxicology, 278(2), 224-228.
Lichtenfels, Rudolf, Dressler, Sven P, Zobawa, Monica, Recktenwald, Christian V, Ackermann,
Angelika, Atkins, Derek, . . . Lottspeich, Friedrich. (2009). Systematic Comparative
98
Protein Expression Profiling of Clear Cell Renal Cell Carcinoma A PILOT STUDY
BASED ON THE SEPARATION OF TISSUE SPECIMENS BY TWO-
DIMENSIONAL GEL ELECTROPHORESIS. Molecular & Cellular Proteomics, 8(12),
2827-2842.
Linge, Annett, Kennedy, Susan, O'Flynn, Deirdre, Beatty, Stephen, Moriarty, Paul, Henry,
Michael, . . . Meleady, Paula. (2012). Differential expression of fourteen proteins
between uveal melanoma from patients who subsequently developed distant metastases
versus those who did not. Investigative ophthalmology & visual science, 53(8), 4634-
4643.
Lorigan, JG, Wallace, S, & Mavligit, GM. (1991). The prevalence and location of metastases
from ocular melanoma: imaging study in 110 patients. AJR. American journal of
roentgenology, 157(6), 1279-1281.
Lu, L., Gu, X., Hong, L., Laird, J., Jaffe, K., Choi, J., . . . Salomon, R. G. (2009). Synthesis and
structural characterization of carboxyethylpyrrole-modified proteins: mediators of age-
related macular degeneration. Bioorg Med Chem, 17(21), 7548-7561. doi:
10.1016/j.bmc.2009.09.009
Michels-Rautenstrauss, Karin G, Mardin, Christian Y, Zenker, Martin, Jordan, Nicole, Gusek-
Schneider, Gabriele-Charlotte, & Rautenstrauss, Bernd W. (2001). Primary congenital
glaucoma: three case reports on novel mutations and combinations of mutations in the
GLC3A (CYP1B1) gene. Journal of glaucoma, 10(4), 354-357.
Mudunuri, K. (2008). Intravitreal Delivery of Corticosteroid Nanoparticles. (Ph.D. Ph.D.),
University of Florida, Gainesville, FL. Retrieved from
http://ufdc.ufl.edu/UFE0022065/00001
NEI. (2012). Prevalence of Adult Vision Impairment and Age-Related Eye Diseases in America.
Retrieved 11/20/13, 2013
Nicolini, C. (2006). Nanogenomics for medicine. Nanomedicine (Lond), 1(2), 147-152. doi:
10.2217/17435889.1.2.147
Ong, F. S., Kuo, J. Z., Wu, W. C., Cheng, C. Y., Blackwell, W. L., Taylor, B. L., . . . Wong, T.
Y. (2013). Personalized Medicine in Ophthalmology: From Pharmacogenetic Biomarkers
to Therapeutic and Dosage Optimization. J Pers Med, 3(1), 40-69. doi:
10.3390/jpm3010040
99
Ong, Frank S, Kuo, Jane Z, Wu, Wei-Chi, Cheng, Ching-Yu, Blackwell, Wendell-Lamar B,
Taylor, Brian L, . . . Wong, Tien Y. (2013). Personalized Medicine in Ophthalmology:
From Pharmacogenetic Biomarkers to Therapeutic and Dosage Optimization. Journal of
Personalized Medicine, 3(1), 40-69.
Park, Y. G., Rhu, H. W., Kang, S., & Roh, Y. J. (2012). New Approach of Anti-VEGF Agents
for Age-Related Macular Degeneration. J Ophthalmol, 2012, 637316. doi:
10.1155/2012/637316
Penfold, P. L., Wen, L., Madigan, M. C., Gillies, M. C., King, N. J., & Provis, J. M. (2000).
Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1
(ICAM-1) expression of the ECV304 cell line: implications for macular degeneration.
Clin Exp Immunol, 121(3), 458-465.
Peyman, G. A., Lad, E. M., & Moshfeghi, D. M. (2009). Intravitreal injection of therapeutic
agents. Retina, 29(7), 875-912. doi: 10.1097/IAE.0b013e3181a94f01
Qian, Xiao-Long, Shi, Qing-Guo, Pang, Bo, Wu, Rui-Qin, Yu, Lan, Li, Shan-Hu, . . . Zhou, Jian-
Guang. (2010). [Identification and expression of two new secretory proteins associated
with prostate cancer]. Yi chuan= Hereditas/Zhongguo yi chuan xue hui bian ji, 32(3),
235-241.
Quigley, Harry A. (1996). Number of people with glaucoma worldwide. British Journal of
Ophthalmology, 80(5), 389-393.
Rainov, Nicolai G. (2000). A phase III clinical evaluation of herpes simplex virus type 1
thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and
radiation in adults with previously untreated glioblastoma multiforme. Human gene
therapy, 11(17), 2389-2401.
Ramasamy, Pathma, Murphy, Conor C, Clynes, Martin, Horgan, Noel, Moriarty, Paul, Tiernan,
Damien, . . . Meleady, Paula. (2014). Proteomics in uveal melanoma. Experimental eye
research, 118, 1-12.
Rosenfeld, P. J., Brown, D. M., Heier, J. S., Boyer, D. S., Kaiser, P. K., Chung, C. Y., . . . Group,
Marina Study. (2006). Ranibizumab for neovascular age-related macular degeneration. N
Engl J Med, 355(14), 1419-1431. doi: 10.1056/NEJMoa054481
100
Samudre, S. S., Lattanzio, F. A., Jr., Williams, P. B., & Sheppard, J. D., Jr. (2004). Comparison
of topical steroids for acute anterior uveitis. J Ocul Pharmacol Ther, 20(6), 533-547. doi:
10.1089/jop.2004.20.533
Selicharova, Irena, Sanda, Miloslav, Miadkova, J, Ohri, Sujata Saraswat, Vashishta, Aruna,
Fusek, Martin, . . . Vetvicka, Vaclav. (2008). 2-DE analysis of breast cancer cell lines
1833 and 4175 with distinct metastatic organ-specific potentials: comparison with
parental cell line MDA-MB-231. Oncology reports, 19(5), 1237-1244.
Sendrowski, D.P., Jaanus, S.D., Semes, L.P., & Stern, M.E. (2008). Anti-Inflammatory Drugs
(5th ed.). New York, NY: Elsevier Health Sciences.
Sherif, Z., & Pleyer, U. (2002). Corticosteroids in ophthalmology: past-present-future.
Ophthalmologica, 216(5), 305-315. doi: 66189
Sigurdsson, H. H., Konraethsdottir, F., Loftsson, T., & Stefansson, E. (2007). Topical and
systemic absorption in delivery of dexamethasone to the anterior and posterior segments
of the eye. Acta Ophthalmol Scand, 85(6), 598-602. doi: 10.1111/j.1600-
0420.2007.00885.x
Singh, Arun D, & Borden, Ernest C. (2005). Metastatic uveal melanoma. Ophthalmology clinics
of North America, 18(1), 143-150, ix.
Singh, Arun D, & Topham, Allan. (2003). Survival rates with uveal melanoma in the United
States: 1973–1997. Ophthalmology, 110(5), 962-965.
Steely, H. T., Jr., & Clark, A. F. (2000). The use of proteomics in ophthalmic research.
Pharmacogenomics, 1(3), 267-280. doi: 10.1517/14622416.1.3.267
Stoilov, I, Jansson, Ingela, Sarfarazi, M, & Schenkman, John B. (2001). Roles of cytochrome
p450 in development. Drug metabolism and drug interactions, 18(1), 33-56.
Stone, Edwin M, Fingert, John H, Alward, Wallace LM, Nguyen, Thai D, Polansky, Jon R,
Sunden, Sara LF, . . . Nichols, Brian E. (1997). Identification of a gene that causes
primary open angle glaucoma. Science, 275(5300), 668-670.
Urtti, A. (2006). Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv
Drug Deliv Rev, 58(11), 1131-1135. doi: 10.1016/j.addr.2006.07.027
Vadlapudi, A.D., Ashaben, P. , Kishore, C., & Ashim, K.M. (2012). Recent Patents on Emerging
Therapeutics for the Treatment of Glaucoma, Age Related Macular Degeneration and
Uveitis. Recent Pat Biomed Eng, 5(1), 83-101.
101
Verteporfin In Photodynamic Therapy Study, Group. (2001). Verteporfin therapy of subfoveal
choroidal neovascularization in age-related macular degeneration: two-year results of a
randomized clinical trial including lesions with occult with no classic choroidal
neovascularization--verteporfin in photodynamic therapy report 2. Am J Ophthalmol,
131(5), 541-560.
Wadhwa, Sheetu, Paliwal, Rishi, Paliwal, Shivani R, & Vyas, SP. (2010). Hyaluronic acid
modified chitosan nanoparticles for effective management of glaucoma: development,
characterization, and evaluation. Journal of drug targeting, 18(4), 292-302.
Wang, Y. S., Friedrichs, U., Eichler, W., Hoffmann, S., & Wiedemann, P. (2002). Inhibitory
effects of triamcinolone acetonide on bFGF-induced migration and tube formation in
choroidal microvascular endothelial cells. Graefes Arch Clin Exp Ophthalmol, 240(1),
42-48.
Wang, Y., Wang, V. M., & Chan, C. C. (2011). The role of anti-inflammatory agents in age-
related macular degeneration (AMD) treatment. Eye (Lond), 25(2), 127-139. doi:
10.1038/eye.2010.196
Wang, Yingchih, Mo, Li, Wei, Wenbin, & Shi, Xuehui. (2013). Efficacy and safety of dendrimer
nanoparticles with coexpression of tumor necrosis factor-α and herpes simplex virus
thymidine kinase in gene radiotherapy of the human uveal melanoma OCM-1 cell line.
International journal of nanomedicine, 8, 3805.
Weinreb, Robert N, Kashiwagi, Kenji, Kashiwagi, Fumiko, Tsukahara, Shigeo, & Lindsey,
James D. (1997). Prostaglandins increase matrix metalloproteinase release from human
ciliary smooth muscle cells. Investigative ophthalmology & visual science, 38(13), 2772-
2780.
Wong, Tina TL, Sethi, Charanjit, Daniels, Julie T, Limb, G Astrid, Murphy, Gillian, & Khaw,
Peng T. (2002). Matrix metalloproteinases in disease and repair processes in the anterior
segment. Survey of ophthalmology, 47(3), 239-256.
Wu, W. S., Wang, F. S., Yang, K. D., Huang, C. C., & Kuo, Y. R. (2006). Dexamethasone
induction of keloid regression through effective suppression of VEGF expression and
keloid fibroblast proliferation. J Invest Dermatol, 126(6), 1264-1271. doi:
10.1038/sj.jid.5700274
102
WuDunn, Darrell. (2002). Genetic basis of glaucoma. Current opinion in ophthalmology, 13(2),
55-60.
Zarbin, Marco Attilio, Montemagno, Carlo, Leary, James Francis, & Ritch, Robert. (2013).
Nanomedicine for the treatment of retinal and optic nerve diseases. Current opinion in
pharmacology, 13(1), 134-148.
Zhang, Li, Cilley, Robert E, & Chinoy, Mala R. (2000). Suppression subtractive hybridization to
identify gene expressions in variant and classic small cell lung cancer cell lines. Journal
of Surgical Research, 93(1), 108-119.
Zhang, W., Prausnitz, M. R., & Edwards, A. (2004). Model of transient drug diffusion across
cornea. J Control Release, 99(2), 241-258. doi: 10.1016/j.jconrel.2004.07.001
Zhao, Xiujun, Ramsey, Keri E, Stephan, Dietrich A, & Russell, Paul. (2004). Gene and protein
expression changes in human trabecular meshwork cells treated with transforming growth
factor-β. Investigative ophthalmology & visual science, 45(11), 4023-4034.
Zhou, Yixiong, Song, Xin, Jia, Renbin, Wang, Haibo, Dai, Liyan, Xu, Xiaofang, . . . Fan,
Xianqun. (2010). Radiation ‐ inducible human tumor necrosis factor ‐ related
apoptosis‐inducing ligand (TRAIL) gene therapy: a novel treatment for radioresistant
uveal melanoma. Pigment cell & melanoma research, 23(5), 661-674.
Zhou, Zhaohui, & Vollrath, Douglas. (1999). A cellular assay distinguishes normal and mutant
TIGR/myocilin protein. Human molecular genetics, 8(12), 2221-2228.
top related